Nucleic acid molecule encoding homer 1B protein

ABSTRACT

A method is provided for identifying a compound that modulates a cellular response associated with Homer and mediated by a cell-surface or an intracellular receptor. A method is further provided for identifying a compound that modulates receptor activated calcium mobilization associated with Homer. A method is provided for identifying a compound that inhibits Homer protein activity based on the crystal structure coordinates of Homer protein binding domain. A method is also provided for identifying a compound that affects the formation of cell surface receptors into clusters. Also provided are nucleic acids encoding Homer proteins as well as Homer proteins, and Homer interacting proteins.

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application No. 60/097,334, filed Aug. 18, 1998, toU.S. Provisional Application No. 60/138,426, filed Jun. 10, 1999, toU.S. Provisional Application No. 60/138,493, filed Jun. 10, 1999, and toU.S. Provisional Application No. 60/138,494, filed Jun. 10, 1999, eachof which is incorporated by reference in its entirety herein.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. RO1DA10309, RO1 DA11742 and KO2 MH01152, awarded by the National Institutesof Health. The government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to protein-protein interactionsand more specifically to molecules involved in mediatingreceptor-activated or ion channel-mediated intracellular calciummobilization or concentration.

BACKGROUND OF THE INVENTION

The mature central nervous system exhibits the capacity to altercellular interactions as a function of the activity of specific neuronalcircuits. This capacity is believed to underlie learning and memorystorage, age-related memory loss, tolerance to and dependence on drugsof abuse, recovery from brain injury, epilepsy as well as aspects ofpostnatal development of the brain (Schatz, C., Neuron, 5:745, 1990).Currently, the role of activity-dependent synaptic plasticity is bestunderstood in the context of learning and memory. Cellular mechanismsunderlying activity-dependent plasticity are known to be initiated byrapid, transmitter-induced changes in membrane conductance propertiesand activation of intracellular signaling pathways (Bliss andCollingridge, Nature, 361:31, 1993). Several lines of evidence alsoindicate a role for rapid synthesis of mRNA and protein in long-termneuroplasticity. For example, classical studies of learning and memorydemonstrate a requirement for protein synthesis in long-term, but notshort-term memory (Flexner, et al., Science, 141:57, 1963; Agranoff, B.,Basic Neurochemistry, 3rd Edition, 1981; Davis and Squire, Physiol.Bull., 96:518, 1984), and long-term enhancement of synapticconnectivity, studied in cultured invertebrate neurons (Montarolo, etal., Science, 234:1249, 1986; Bailey, et al., Neuron, 9:749, 1992) or inthe rodent hippocampus (Frey, et al., Science, 260:1661, 1993; Nguyen,et al., Science, 265:1104, 1994), is blocked by inhibitors of either RNAor protein synthesis. Importantly, inhibitors of macromolecularsynthesis are most effective when administered during a brief timewindow surrounding the conditioning stimulus indicating a specialrequirement for molecules that are rapidly induced (Goelet, et al.,Nature, 322:419, 1986).

Immediate early genes (IEGs) are rapidly induced in neurons byneurotransmitter stimulation and synaptic activity and are hypothesizedto be part of the macromolecular response required for long-termplasticity (Goelet, et al., supra; Sheng and Greenberg, Neuron, 4:477,1990; Silva and Giese, Neurobiology, 4:413, 1994). To identify cellularmechanisms that may contribute to long-term plasticity in the vertebratebrain, differential cloning techniques have been used to identify genesthat are rapidly induced by depolarizing stimuli (Nedivi, et al.,Nature, 363:713, 1993; Qian, et al., Nature, 361:453, 1993; Yamagata, etal., Neuron, 11:371, 1993; Yamagata, et al., Learning and Memory 1:140,1994; Yamagata, et al., Journal of Biological Chemistry, 269:16333,1994; Andreasson and Worley, Neuroscience, 69:781, 1995; Lyford, et al.,Neuron, 14:433, 1995). In contrast to the earlier focus on transcriptionfactors, many of the newly characterized IEGs represent molecules thatcan directly modify the function of cells and include growth factors(Nedivi, et al., supra; Andreasson and Worley, supra), secreted enzymesthat can modify the extracellular matrix, such as tissue plasminogenactivator (Qian, et al., supra), enzymes involved in intracellularsignaling, such as prostaglandin synthase (Yamagata, et al., supra), anda novel homolog of H-Ras, termed Rheb (Yamagata, et al., supra), as wellas a novel cytoskeleton-associated protein, termed Arc (Lyford, et al.,supra). The remarkable functional diversity of this set of rapidresponse genes is representative of the repertoire of cellularmechanisms that are likely to contribute to activity-dependent neuronalplasticity.

Pharmaceutical agents often act by modulating signaling between cells orwithin cells. For example, Prozac alters the reuptake of theneurotransmitter serotonin and enhances aspects of its signalingfunction in brain. Nonsteroidal antiinflammatory drugs (NSAIDs) act byinhibiting the activity of cyclooxygenase enzyme, which is involved inthe signaling pathways of inflammation. Viagra modifies theintracellular guanylate cyclase response to autonomic neurotransmittersin erectile tissues. These, and other precedent setting pharmaceuticals,validate the notion that specific signaling pathways may be targeted fortherapeutic development.

Cellular mechanisms that modify important intracellular signals caninvolve changes in intracellular calcium. This type of mechanism is usedin brain neurons to adapt to changes in intercellular signaling, and isdemonstrated to exert powerful effects on cellular responses induced byglutamate. Similar, though distinct, cellular mechanism may be used tomodulate intracellular calcium signals in other tissues including heart,lung, liver and skeletal muscle. Compounds that can modify thismechanism can modulate natural transmitter signals and may exerttherapeutic effects.

Classical studies demonstrated that activation of receptors on the cellsurface evoke changes in the level of specific, diffusable moleculesinside the cell. The regulated production of these molecules serves tosignal events happening at the membrane surface to intracellularreceptors and are therefore termed second messenger signaling pathways.Major second messenger pathways include the phosphoinositide pathway,which regulates intracellular calcium; the adenylate cyclase pathway,which regulates levels of cyclic AMP; the guanylate cyclase pathway,which regulates levels of cGMP; and the nitric oxide pathway whichregulates NO.

The regulated release of intracellular calcium is essential to thefunction of all tissues. Each tissue possesses a distinct physiologythat is dependent on receptor/transmitter-regulated release ofintracellular calcium. For example, synaptic function is modulated inbrain neurons by glutamate receptor regulated release of intracellularcalcium. Contractility of cardiac and smooth muscle is also regulated byintracellular calcium. Recent reviews of the role of calcium signalingin cellular responses include: Berridge, Nature 386:759 (1997);Berridge, J. Physiol. (London) 499:291 (1997); Bootman et al., Cell91:367 (1997).

Recent studies demonstrate that molecules that function together insignaling networks are frequently clustered together in macromolecularcomplexes. For example, components of the MAP kinase pathway form acomplex of cytosolic kinases with their specific substrates (Davis, Mol.Reprod. Dev. 42:459 (1995)). Similarly, proteins such as AKAP functionas scaffolds for specific kinases and their substrates (Lester andScott, Recent Prog. Horm. Res. 52:409 (1997)). Recently, a multi-PDZcontaining protein was identified in Drosophila (termed InaD) thatcouples the membrane-associated, light-activated ion channel with itseffector enzymes (Tsunoda et al., Nature 388:243 (1997)). Thebiochemical consequence of this clustering is that the localconcentrations of molecules that convey the signals between proteins areas high as possible. Consequently, signaling takes place efficiently.The clustering activity of these proteins is essential to normalfunction of the signaling cascade (Lester and Scott, supra 1997; Tsunodaet al., supra 1997). Accordingly, Accordingly, agents that alter thesesignaling complexes will modify the response due to transmitter or otherform of cellular stimulation in a way that mimics more classicalreceptor agonists or antagonists. For example, a metabotropic glutamatereceptor signaling may be blocked either at the receptor by conventionalreceptor antagonists or by uncoupling the metabotropic receptor from itsintracellular IP3 receptor by agents that block the cross-linkingactivity of Homer family proteins.

The identification of molecules regulating the aggregation ofneurotransmitter receptors at synapses is central to understanding themechanisms of neural development, synaptic plasticity and learning. Themost well characterized model for the synaptic aggregation of ionotropicreceptors is the neuromuscular junction. Early work showed that contactbetween the axon of a motor neuron and the surface of a myotube rapidlytriggers the accumulation of preexisting surface acetylcholine receptors(Anderson and Cohen, J Physiol 268:757-773, 1977; Frank and Fischbach, JCell Biol 83:143-158, 1979). Subsequent work has shown that agrin, acomplex glycoprotein secreted by the presynaptic terminal, activates apostsynaptic signal transduction cascade (reviewed by Colledge andFroehner, Curr Opin Neurobiol 8:357-63, 1998), that leads to receptorclustering by the membrane associated protein rapsyn.

SUMMARY OF THE INVENTION

Homer proteins, the products of neuronal immediate early genes,selectively bind the carboxy-termini of certain cell-surface receptors(e.g., group 1 metabotropic receptors), certain intracellular receptorsand binding proteins (e.g., inositol trisphosphate receptors, ryanodinereceptor, Shank proteins, I42). Many forms of Homer proteins contain a“coiled-coil” structure in the carboxy-terminal domain which mediateshomo- and heteromultimerization between Homer proteins. The presentinvention is based on the seminal discovery that Homer plays asignificant role in mediating receptor-activated calcium mobilizationfrom internal stores and that Homer proteins regulate aspects ofreceptor clustering

In one embodiment, a method is provided for identifying a compound thatmodulates a cellular response mediated by a cell-surface receptor. Themethod includes incubating a test compound and a cell expressing acell-surface receptor and a Homer protein under conditions sufficient topermit the compound to interact with the cell, and exposing the cell toa cell-surface receptor ligand. A cellular response to the ligand by thecell incubated with the compound is compared with a cellular response ofthe cell not incubated with the compound wherein a difference incellular response identify a compound that modulates a Homer-associatedcellular response.

In another embodiment, a method is provided for identifying a compoundthat modulates a cellular response mediated by an intracellularreceptor. The method includes incubating the compound, and a cellexpressing an intracellular receptor and a Homer protein underconditions sufficient to permit the compound to interact with the celland exposing the cell to conditions that activate the intracellularreceptor. A cellular response by a cell incubated with the compound iscompared with a cellular response of a cell not incubated with thecompound wherein a difference in a cellular response identifies acompound that modulates a Homer-associated cellular response.

In yet another embodiment, a method is provided for identifying acompound that modulates receptor activated calcium mobilization in acell. The method includes incubating the compound and a cell expressinga Homer protein under conditions sufficient to permit the compound tointeract with the cell and exposing the cell to conditions sufficient toactivate calcium mobilization. The receptor-activated calciummobilization of a cell incubated with said the compound is compared withthe receptor-activated calcium mobilization of a cell not incubated withthe compound wherein a difference in calcium mobilization is indicativeof an effect of the compound on Homer-associated calcium mobilization.

In another embodiment, a method is provided for modulatingreceptor-mediated calcium mobilization. The method includes exposing acell expressing Homer protein to a compound in a sufficient amount tomodulate the calcium mobilization that typically occurs when a cell isexposed to an amount of ligand sufficient to activate an intercellularsignaling pathway that includes Homer protein.

In another embodiment, a method is provided for identifying a compoundthat inhibits Homer protein activity. The method includes identifying aninhibitor of Homer binding or crosslinking activity and identifying aninhibitor of Homer protein activity that forms covalent or non-covalentbonds with amino acids in a Homer protein binding site, based upon thecrystal structure coordinates of Homer protein binding domain. andsynthesizing the inhibitor.

In one embodiment, a method is provided for identifying a compound thataffects the formation of cell surface receptors into clusters. Themethod includes incubating the compound and a cell expressing a Homerprotein and a Homer interacting protein, e.g., a Shank protein, underconditions sufficient to allow the compound to interact with the celland determining the effect of the compound on the formation ofcell-surface receptors into clusters. The formation of cell-surfacereceptors into clusters of a cell contacted with the compound iscompared to the formation of cell-surface receptors into clusters of acell not contacted with the compound, wherein a difference in theformation of clusters is indicative of a compound that affects formationof cell surface receptors into clusters.

In another embodiment, a method is provided for treating a disorderassociated with glutamate receptors, including metabotropic andNMDA-type glutamate receptors, in a subject. The method includesadministering to a subject in need, a therapeutically effective amountof a compound that modulates Homer protein activity.

In another embodiment, a method is provided for treating a disorderassociated with Homer protein activity including administering to asubject in need a therapeutically effective amount of a compound thatmodulates Homer protein activity. The compound may be identified by amethod of the invention described herein.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 1b, having the nucleotide sequence as set forthin SEQ ID NO:3 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:4.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 1c, having the nucleotide sequence as set forthin SEQ ID NO:5 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:6.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 2a, having the nucleotide sequence as set forthin SEQ ID NO:7 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:8.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 2b, having the nucleotide sequence as set forthin SEQ ID NO:9 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:10.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 3, having the nucleotide sequence as set forth inSEQ ID NO:11 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:12.

In another embodiment, there is provided an isolated peptide having theamino acid sequence set forth in SEQ ID NO:13 an isolated peptide havingthe amino acid sequence set forth in SEQ ID NO:14.

In yet another embodiment, there is provided an isolated nucleic acidencoding Homer Interacting Protein, having the nucleotide sequence asset forth in SEQ ID NO:15 or 17 with a deduced amino acid sequence asset forth in SEQ ID NO:16 or 18, respectively.

In another embodiment, there is provided an isolated Homer InteractingProtein having substantially the same amino acid sequence as set forthin SEQ ID NO:19.

In another embodiment, there is provided an isolated Homer InteractingProtein having substantially the same amino acid sequence as set forthin SEQ ID NO:20.

In yet a further embodiment, there is provided a substantially purifiedpolypeptide containing a proline rich region that is specificallycapable of specifically binding to polypeptides of the Homer family.

In still another embodiment, there is provided a transgenic non-humananimal having a transgene that expresses a Homer protein, e.g., Homer1a, chromosomally integrated into the germ cells of the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic Representation of EVH1 Domain-containing Proteins.EVH1 domains are found at or near the N-termini of Homer, Ena, Mena,VASP, and WASP proteins. Homer 1b/2/3 encode a CC domain which mediatesmultimerization between various Homer proteins. In ENA, Mena, VASP,WASP, and N-WASP, the EVH1 domain is followed by a central proline richregion of variable length. The proteins are drawn to the scale shown,and the respective amino acid lengths are shown at the right.

FIG. 2. Structure-Based Alignment of EVH1, PH, and PTB Domain Sequences.A structure-based sequence alignment between EVH1 domain and theβ-spectrin PH domain and the IRS-1 PTB domain is shown. Species areindicated by Rn (rat), Hs (human), Mm (mouse), and Dm (Drosophila).Elements of the Homer EVH1 domain secondary structure are represented byarrows (β-strands), cylinders (α-helices), and lines (coils). Conservedresidues (among EVH1 domains) are highlighted. The fractional solventaccessibility (FAS) of each residue in Homer 1a is indicated by ovals.Filled ovals=0≦FAS≦0.1 (buried); shaded ovals=0.1<FAS≦0.4 (partiallyaccessible); open ovals=FAS>0.4. Mutations in the EVH1 domain of theWASP gene are indicated in lower case letter below the WASP amino acidsequence. Mutations that are associated with the severe WAS phenotypeare show in bold letters (Zhu et al, 1997). Sites mutated to more thenone residue are indicated by asterisks. Bold asterisk indicate residuesthat, when mutated, affect the interaction of WASP with WIP (Stewart etal., 1999). Residues of Homer, β-spectrin, and IRS-1 that align wellfollowing structural superposition and were used to calculate rmsdifferences in Cα positions between these domains are underlined in theIRS-1 sequence. Gaps are indicated by dashes while continued sequencesat amino- and carboxy-termini are indicated by periods. Residuenumbering for Homer 1a is shown above its amino acid sequence. Thenumber of the last included residue of each protein is shown it the endof each row. Sequences shown are Homer 1a Rn (SEQ ID NO:63), Homer Dm(SEQ ID NO:64), Ena Dm (SEQ ID NO:65), Mena Mm (SEQ ID NO:66), EVL Mm(SEQ ID NO:67), SIF Dm (SEQ ID NO:68), VASP Hs (SEQ ID NO:69), N-WASP Hs(SEQ ID NO:70), WASP Hs (SEQ ID NO:71), WASP mut (SEQ ID NO:72), β-specMm (SEQ ID NO:6), IRS-1 Hs (SEQ ID NO:5).

FIG. 3. Ribbon Diagram of the Homer 1a EVH 1 Domain. The amino andcarboxy termini are indicated, and elements of secondary structure arelabeled to correspond to homologous structures in PH and PTB domains. Anadditional short region of β-strand between β1 and β2 has been labeledβi.

FIG. 4. Structural Comparison of EVH1, PH, and PTB Domains. Ribbondiagrams (A)-(C) and surface representations (D)-(F) of the Homer 1EVH1, β-spectrin PH, and IRS-1 PTB domains, respectively, are shown. Allmolecules are shown in a similar orientation, which is rotated about 45°about the vertical axis from orientations shown in FIG. 3. Theβ-spectrin PH domain is shown with bound inositol trisphosphate (Hyvonenet al, 1995). The IRS-1 domain is shown complexed to aphosphotyrosine-containing peptide derived from the insulin receptor(ECk et al., 1996).

FIG. 5. Versatile Ligand Recognition by PH-Like Domain. Sterodiagram ofa backbone trace of Homer 1 EVH1 doamin showing the relative positionsof IP3 as bound by the β-spectrin and PLC-δ PH domains, as well as thepeptide ligands for the IRS-1 and Numb PTB domains is shown. Theorientations of the EVH1 domain is similar to that in FIG. 4. Ligandpositions were determined by superimposing the backbone traces of theEVH1, PH and PTB domains in the program) (Jones et al., 1991).

FIG. 6. Mapping of WAS-Causing and Homer Binding Mutations on the EVH1Surface. (A) and (B) Surface representations of the Homer1 EVH1 doaminwith sites homologous to positions of WASP mutations (in parentheses)colored according to solvent accessibility. Solvent exposed residues areshown in magenta, and buried or partially buried residues are shown inblue. Residue assignments are based on the sequence shown in FIG. 2.WASP EVH1 mutations are listed in Table 2. Surface representations ofHomer 1 EVH1 domain showing the location of residues targeted bysite-directed mutagenesis. Mutations that disrupt binding of Homer EVH1to ligands in an in vitro binding assay are shown in red, while thosethat have no effect on binding are shown in light blue (see Table 3).The orientation of the EVH1 domain in panels A and C is identical tothat in FIG. 4A and D. IN panels B and D, the molecule is rotated about180 degrees about the vertical axis.

FIGS. 7 through 45 are described in the following table.

Figures Homer Family Proteins and Homer Interacting Proteins FIG. SEQ IDNo. No. Sequence 1 Human Homer 1a (nucleic acid) 7 2 Human Homer 1a(amino acid) 8 3 Human Homer 1b (nucleic acid) 9 4 Human Homer 1b (aminoacid) 2 5 IRS-1 2 6 β-spectrin 10 7 Human Homer 2a (nucleic acid) 11 8Human Homer 2a (amino acid) 12 9 Human Homer 2b (nucleic acid) 13 10Human Homer 2b (amino acid) 14 11 Human Homer 3 (nucleic acid) 15 12Human Homer 3 (amino acid) 16 15 Homer interacting protein: rat I30(nucleic acid) 17 16 Homer interacting protein: rat I30 (amino acid)18-1 to 17 Homer interacting protein: rat I42 (nucleic acid) 18-2 19 18Homer interacting protein: rat I42 (amino acid) 20 19 Homer interactingprotein: human I30 (nucleic acid) 21 20 Homer interacting protein: humanI30 (amino acid) 22-1 to 21 Homer interacting protein: human I42 22-3(nucleic acid) 23 22 Homer interacting protein: human I42 (amino acid)24 23 Mouse Homer 1a (nucleic acid) 25 24 Mouse Homer 1a (amino acid) 2625 Mouse Homer 1b (nucleic acid) 27 26 Mouse Homer 1b (amino acid) 28 27Mouse Homer 2a (nucleic acid) 29 28 Mouse Homer 2a (amino acid) 30 29Mouse Homer 2b (nucleic acid) 31 30 Mouse Homer 2b (amino acid) 32 31Mouse Homer 3 (nucleic acid) 33 32 Mouse Homer 3 (amino acid) 34-1 to 33Rat Homer 1a (nucleic acid) 34-3 35 34 Rat Homer 1a (amino acid) 36-1 to35 Rat Homer 1b (nucleic acid) 36-2 37 36 Rat Homer 1b (amino acid) 38-1to 37 Rat Homer 1c (nucleic acid) 38-2 39 38 Rat Homer 1c (amino acid)40-1 to 39 Rat Shank 3a (nucleic acid) 40-4 41 40 Rat Shank 3a (aminoacid) 42 41 Human Homer 3a (nucleic acid) 43 42 Human Homer 3a (aminoacid) 44 43 Rat INADL partial nucleic acid sequence 45 44 Rat INADLpartial amino acid sequence

DETAILED DESCRIPTION OF THE INVENTION

Homer represents a family of proteins that selectively binds thecarboxy-terminus of group 1 metabotropic receptors and is enriched atexcitatory synapses (Brakeman et al., 1977). In the adult brain, Homeris rapidly and transiently induced by physiological synaptic stimulithat evoke ion-term potentiation in the hippocampus (Brakeman et al.,1997; Kato et al., 1997), and is also induced in the striatum bydopaminetic drugs of addiction (Brakeman et al., 1997). The first Homergene identified, now termed Homer 1a (Brakeman et al., Nature386:2284-288 (1997); GenBank Accession No. U92079), is a member of afamily of closely related Homer proteins that are constitutivelyexpressed in brain (Kato et al., 1998; Sun et al., 1998; Xiao et al.,1998). There are now three mammalian genes identified and at least sixdistinct transcripts expressed in brain (Xiao et al., 1998). All Homerfamily members, including Homer 1a, contain an amino-terminal region ofabout 110 amino acids that binds metabotropic glutamate receptors 1a and5 (mGluR1a and mGluR5) (Xiao et al., 1998). The region of Homer thatinteracts with mGluR1a or 5 is termed “EVH1 domain”, based on homologyto similar domains in a family of proteins that include DrosophilaEnabled (Gertler et al., 1996), mammalian VASP (Haffner et al., 1995)and the Wescott-Aldrige protein (WASP) (Ponting and Phillips, 1997;Symons et al., 1996). The EVH1 domain of Homer is conserved at a levelof about 80% between Drosophila, rodent and human (Xiao et al., 1998)The Homer family EVH1 domain also can bind to intracellular receptorssuch as the inositol trisphosphate receptor and dyamin III. Binding ofHomer proteins in the EVH1 region is mediated by an amino acid sequencemotif that is rich in proline residues.

To explore the proline-rich motif and its role in Homer interactions, adeletion mutation strategy was used. A 50-amino acid deletion at thecarboxy-terminal end of mGluR5 destroyed binding to Homer. By contrast,a 41 amino acid deletion of mGluR5 retained full binding activity. Theintervening sequence is proline rich and shares sequence similarity withthe previously described SH3 ligand sequence (Yu, 1994 ). A series ofpoint mutants based on the known structure-function relationship for SH3ligands was prepared and binding assays confirmed generalcharacteristics of SH3 ligand binding, but also demonstrated that thatthe Homer binding site is distinct in the positioning of critical aminoacids (Tu et al., 1998). A consensus for binding was determined to bePPXXFR, consistent with the observation that mutation of either of theproline residues or the phenylalanine, or a change in their relativeposition, interrupted binding. The arginine in the last position waspreferred over other tested amino acids, but is not essential. Mutationswere identically effective in interrupting binding to each of the Homerfamily members including Homer 1a, 1b/c, 2a/b, 3 and an EVH1 fragment(110 amino acids) of Homer 1. Thus, it was concluded that theinteraction with mGluR5 was mediated by the Homer EVH1 domain.

To further explore Homer binding, mutations of mGluR5 were tested usinga 250 amino acid carboxy-terminal fragment of the receptor, which had anidentical effect on binding when placed in the full length mGluR5protein (Tu et al., 1998). This exquisite sensitivity of Homer bindingto changes in single amino acids within the Homer-ligand site wasconfirmed in other Homer-interacting proteins including mGluR1a (Tu etal., 1998), Shank (Tu et al., 1999), and 142 (see below). To furtherconfirm that the interaction was mediated by a direct interaction at theHomer-ligand site (as opposed to a secondary allosteric effect on aremote binding site), synthetic 10-mer peptides with either the wildtype, or F-to-R mutation were prepared. The wild type peptide blockedbinding of mGluR1a or mGluR5 to each of the Homer family members (Tu etal., 1998). Approximately half of the binding was blocked at a peptideconcentration of 3.4 micromolar. By contrast, the F-to-R mutant peptidedid not alter binding at concentrations as high as 340 micromolar.

Most forms of Homer protein encode a carboxy-terminal domain with a“coiled-coil” structure. This coiled-coil domain mediates homo- andheteromultermization between Homer proteins (Kato et al., 1998; Xiao etal., 1998) and such multimers can be identified in normal brain tissue(Xiao et al., 1998). Homer proteins are enriched in brain tissuefractions from postsynaptic densities and are localized at theultrastructural level to postsynaptic densities. Homer 1a differs fromthe other members of the Homer family in that Homer 1a is notconstitutively expressed and it does not contain a carboxy terminalcoiled-coil domain. Experimental data showing that Homer proteinsinteract with cell-surface receptors and with intracellular receptors,and form multimeric complexes with other Homer proteins indicates animportant role for Homer proteins in intracellular signaling.

An exemplary polynucleotide encoding a Homer protein is set forth as SEQID NO: 1. The term “polynucleotide”, “nucleic acid”, “nucleic acidsequence”, or “nucleic acid molecule” refers to a polymeric form ofnucleotides at least 10 bases in length. By “isolated polynucleotide” ismeant a polynucleotide that is not immediately contiguous with both ofthe coding sequences with which it is immediately contiguous (one on the5′ end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides of the inventioncan be ribonucleotides, deoxyribonucleotides, or modified forms ofeither nucleotide. A polynucleotide encoding Homer includes “degeneratevariants”, sequences that are degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequences areincluded in the invention as long as the amino acid sequence of apolypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 isfunctionally unchanged.

A nucleic acid molecule encoding Homer includes sequences encodingfunctional Homer polypeptides as well as functional fragments thereof.As used herein, the term “functional polypeptide” refers to apolypeptide which possesses biological function or activity which isidentified through a defined functional assay (e.g., EXAMPLE 3), andwhich is associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The term “functional fragments ofHomer polypeptide,” refers to fragments of a Homer polypeptide thatretain a Homer activity, e.g., the ability to interact with cell-surfaceor intracellular receptors or mediate intracellular calciummobilization, and the like. Additionally, functional Homer fragments mayact as competitive inhibitors of Homer binding, for example,biologically functional fragments, for example, can vary in size from apolypeptide fragment as small as an epitope capable of binding anantibody molecule to a large polypeptide capable of participating in thecharacteristic induction or programming of phenotypic changes within acell.

A functional Homer polypeptide includes a polypeptide as set forth inSEQ ID NO:2 and conservative variations thereof. The terms “conservativevariation” and “substantially similar” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like. The terms“conservative variation” and “substantially similar” also include theuse of a substituted amino acid in place of an unsubstituted parentamino acid provided that antibodies raised to the substitutedpolypeptide also immunoreact with the unsubstituted polypeptide.

Also included are other Homer nucleic acid and amino acid sequences,including Homer 1b (SEQ ID NOS:3 and 4); Homer 1c (SEQ ID NOS:5 and 6);Homer 2a (SEQ ID NOS:7 and 8); Homer 2b (SEQ ID NOS:9 and 10); Homer 3(SEQ ID NOS:11 and 12).

Cell-surface receptors are important intermediaries in intercellularsignaling. A “cell-surface receptor” is a protein, usually having atleast one binding domain on the outer surface of a cell where specificmolecules may bind to, activate, or block the cell surface receptor.Cell surface receptors usually have at least one extracellular domain, amembrane spanning region (“transmembrane”) and an intracellular domain.Activation of a cell-surface receptor can lead to changes in the levelsof various molecules inside the cell. Several types of cell-surfacereceptors have been identified in a variety of cell types, includingligand-gated receptors, ligand-gated channels, voltage-activatedreceptors, voltage-activated channels, ion channels and the like.

One class of cell-surface receptor is excitatory amino acid receptors(EAA receptors) which are the major class of excitatory neurotransmitterreceptors in the central nervous system. “EAA receptors” are membranespanning proteins that mediate the stimulatory actions of glutamate andpossibly other endogenous acidic amino acids. EAA are crucial for fastexcitatory neurotransmission and they have been implicated in a varietyof diseases including Alzheimer's disease, stroke schizophrenia, headtrauma and epilepsy. EAA have also been implicated in the process ofaging In addition, EAA are integral to the processes of long-termpotentiation, one of the synaptic mechanisms underlying learning andmemory. There are three main subtypes of EAA receptors: (1) themetabotropic or trans ACPD receptors; (2) the ionotropic NMDA receptors;and (3) the non-NMDA receptors, which include the AMPA receptors andkainate receptors.

Ionotropic glutamate receptors are generally divided into two classes:the NMDA and non-NMDA receptors. Both classes of receptors are linked tointegral cation channels and share some amino acid sequence homology.GluR1-4 are termed AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionicacid) receptors because AMPA preferentially activates receptors composedof these subunits, while GluR5-7 are termed kainate receptors as theseare preferentially sensitive to kainic acid. Thus, an “AMPA receptor” isa non-NMDA receptor that can be activated by AMPA. AMPA receptorsinclude the GluR1-4 family, which form homo-oligomeric andhetero-oligomeric complexes which display different current-voltagerelations and Ca²⁺ permeability. Polypeptides encoded by GluR1-4 nucleicacid sequences can form functional ligand-gated ion channels. An AMPAreceptor includes a receptor having a GluR1, GluR2, GluR3 or GluR4subunit. NMDA receptor subtypes include class NR2B and NR2D, forexample.

Metabotropic glutamate receptors are divided into three groups based onamino acid sequence homology, transduction mechanism and bindingselectivity: Group I, Group II and Group III. Each Group of receptorscontains one or more types of receptors. For example, Group I includesmetabotropic glutamate receptors 1 and 5 (mGluR1 and mGluR5), Group IIincludes metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3)and Group III includes metabotropic glutamate receptors 4, 6, 7 and 8(mGluR4, mGluR6, mGluR7 and mGluR8). Each mGluR type may be found inseveral subtypes. For example, subtypes of mGluR1 include mGluR1a,mGluR1b and mGluR1c.

Group I metabotropic glutamate receptors represent a family of sevenmembrane spanning proteins that couple to G-proteins and activatephospholipase C (Nakanishi, 1994). Members of the family include mGluR1and mGluR5. Activation of these receptors results in the hydrolysis ofmembrane phosphatidylinositol bisphosphate to diacylglycerol, whichactivates protein kinase C. and inositol trisphosphate, which in turnactivates the inositol trisphosphate receptor to release intracellularcalcium. (Aramori and Nakanishi, 1992; Joly et al., 1995 Kawabata etal., 1998)

Activation of a glutamate receptor on the cell surface results in acellular response. A “cellular response” is an event or sequence ofevents that singly or together are a direct or indirect response by acell to activation of a cell surface receptor. A “cellular response” isalso the blockade or activation of selective and non-selective cationchannels and potentiation or inhibition of other cell-surface receptorresponses. In addition, a “cellular response” may be the activation ofan intracellular signaling pathway, including the activation of allsteps or any one step in an intracellular signaling pathway.

An “intracellular signaling pathway” is a sequence of events thattransduces information about an extracellular event into a signal tointracellular receptors or effector molecules such as enzymes. One typeof intracellular signaling pathway is a second messenger signalingpathway. It may begin with the activation of receptors on the cellsurface, which activation evokes changes in the level of specific,diffusible molecules inside the cell. The regulated production of thesemolecules serves to signal events to the intracellular receptors and istherefore termed a second messenger signaling pathway. Major secondmessenger pathways include the adenylate cyclase pathway, whichregulates levels of cyclic AMP, the phosphoinositide pathway, whichregulates intracellular calcium, guanylate cyclase, which regulateslevels of cGMP, and the nitric oxide pathway, which regulates nitricoxide.

A cellular response mediated by cell surface receptors can also includecalcium mobilization. A compound can modulate cellular responsesmediated by cell surface receptors by inhibiting or potentiating therelease of calcium from intracellular stores. A compound increasescalcium mobilization by increasing the release of calcium fromintracellular stores. A compound decreases calcium mobilization byinhibiting of the release of calcium from intracellular stores.

Cell-surface receptors are known to mediate cellular responses. Methodsfor demonstrating cellular responses are well known in the art (e.g.electrophysiological and biochemical methods). (See Examples section foradditional methodology). A method is provided for identifying a compoundthat modulates a cellular response mediated by a cell-surface receptor.The method includes incubating the compound and a cell expressing acell-surface receptor and a Homer protein under conditions sufficient topermit the compound to interact with the cell. The cell may be any cellof interest, including but not limited to neuronal cells, glial cells,cardiac cells, bronchial cells, uterine cells, testicular cells, livercells, renal cells, intestinal cells, cells from the thymus and spleen,placental cells, endothelial cells, endocrine cells including thyroid,parathyroid, pituitary and the like, smooth muscle cells and skeletalmuscle cells. The cell is exposed to a cell-surface receptor ligand. A“cell surface receptor ligand” is a compound that binds to the bindingsite of the cell-surface receptor thereby initiating a sequence ofevents that singly or together embrace a “cellular response”. The effectof the compound on the cellular response is determined, either directlyor indirectly, and a cellular response is then compared with a cellularresponse of a control cell. A suitable control includes, but is notlimited to, a cellular response of a cell not contacted with thecompound. The term “incubating” includes conditions which allow contactbetween the test compound and the cell of interest. “Contacting” mayinclude in solution or in solid phase.

Compounds which modulate a cellular response can include peptides,peptidomimetics, polypeptides, pharmaceuticals, chemical compounds andbiological agents, for example. Antibodies, neurotropic agents,anti-epileptic compounds and combinatorial compound libraries can alsobe tested using the method of the invention. One class of organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 Daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups.

The test agent may also be a combinatorial library for screening aplurality of compounds. Compounds such as peptides identified in themethod of the invention can be further cloned, sequenced, and the like,either in solution of after binding to a solid support, by any methodusually applied to the isolation of a specific DNA sequence Moleculartechniques for DNA analysis (Landegren et al., Science 242:229-237,1988) and cloning have been reviewed (Sambrook et al., MolecularCloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Plainview, N.Y., 1998, herein incorporated by reference).

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc., to producestructural analogs. Candidate agents are also found among biomoleculesincluding, but not limited to: peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

A variety of other agents may be included in the screening assay. Theseinclude agents like salts, neutral proteins, e.g., albumin, detergents,etc. that are used to facilitate optimal protein-protein binding and/orreduce nonspecific or background interactions. Reagents that improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, antimicrobial agents and the like may be used. The mixtureof components are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 10 h will besufficient.

In another embodiment, a method is provided for identifying a compoundthat modulates a cellular response mediated by an intracellularreceptor. An “intracellular receptor” is a protein that binds particularintracellular molecules. Intracellular receptors include ryanodinereceptors and inositol trisphosphate receptors, for example, an“inositol trisphosphate receptor” is a receptor that binds the compoundinositol 1,4,5 trisphosphate, which is an important intracellular secondmessenger. Inositol 1,4,5 trisphosphate is released from phosphatidylinositol bisphosphate by the action of a specific phospholipase C enzyme(PLC) and binds to and activates a calcium channel in the endoplasmicreticulum (ER).

A compound can modulate a cellular response mediated by an intracellularreceptor by inhibiting or potentiating the release of calcium fromintracellular stores, for example, a compound increases calciummobilization by increasing the release of calcium from intracellularstores. A compound decreases calcium mobilization by inhibiting of therelease of calcium from intracellular stores.

The method of the invention includes incubating the compound and a cellexpressing an intracellular receptor and a Homer protein underconditions sufficient to permit the compound to interact with the cell,exposing the cell to conditions that activate said intracellularreceptor, and comparing a cellular response in a cell incubated withsaid compound with the response of a cell not incubated with saidcompound. Methods for determining cellular responses mediated byintracellular signals are well known to one of skill in the art (e.g.,biochemical assays) and provided in the Examples as well.

A method is also provided for identifying a compound that modulatesreceptor-activated calcium mobilization. The term “calcium mobilization”means a change in the amount or concentration of free calcium (Ca⁺²)sequestered in the endoplasmic reticulum, sarcoplasmic reticulum ormitochondria of a cell. The method includes incubating the compound anda cell expressing a Homer protein under conditions sufficient to permitthe compound to interact with the cell and exposing the cell toconditions sufficient to activate calcium mobilization. Then, thecellular response of the cell exposed to the compound is compared to thecellular response of a cell not exposed to the compound. A difference ina cellular response is indicative of a compound that modulatesreceptor-activated calcium mobilization in a cell.

In another embodiment of the invention, a method is provided formodulating receptor-mediated calcium mobilization in a cell includingexposing a cell to a compound in a sufficient amount to modulate thecalcium mobilization that normally occurs when a cell is exposed to allamount of ligand sufficient to activate an intracellular signalingpathway. Those of skill in the art will understand that “the calciummobilization that normally occurs” depends on the cell type and on theligand activating the intracellular pathway (Berridge, 1997 supra;Berridge, 1998 supra; Bootman, 1997 supra). Methods of measuring freecalcium flux are well known in the art (e.g., imaging methodology usingcalcium-sensitive dyes such as fura-2 and the like).

A ligand which activates the intracellular signaling pathway may be anagonist or antagonist of metabotropic glutamate receptors. The terms“agonist” and “antagonist” are meant to include compounds that bind tothe receptor and, respectively, activate or block activation of thereceptor. Known agonists of metabotropic glutamate receptors includeglutamate, quisqualate, Ibotenate, homocysteine sulfinate and theneurotoxin β-N-methylamino-L-alanine. Antagonists of metabotropicglutamate receptors include MCPG. Known agonists of the NMDA typeglutamate receptor include glutamate and NMDA and known antagonistsinclude MK-801 and APV.

Another embodiment of the invention includes a method of identifying acompound that inhibits Homer protein activity. The method relies onfunctional properties of the Homer EVH1 and coiled-coil binding domainsthat can be used to establish high-throughput screens for molecules thatinfluence these and other functional properties of Homer family members.Homer protein activity may be blocked, partially or completely, byinterfering with a protein or other molecule in the intracellularsignaling pathway though which Homer proteins act. For example, Homeractivity can be modulated, for example, by modulating Homer proteinexpression, by modifying the activity of the Homer EVH1 domain, bymodification of the activity of the Homer CC domain, by modification ofHomer crosslinking activity, and the like. Homer activity can also bemodulated with by interfering with the expression or activity of HomerInteracting Protein 142, Homer Interacting Protein 130, NR2D, ACK-2,Shank proteins, ryanodine, inositol trisphosphate, and hInaD, and thelike.

Homer proteins function as a regulated adapter network that cross-linksinteracting proteins. Cross-linking is determined by the bindingproperties of the Homer EVH1 domain, which recognize a uniqueproline-rich ligand with a core sequence consensus of PXXF. This Homerligand is present in all identified proteins that naturally associatewith Homer, and the ability of Homer proteins to bind can be disruptedby single amino acid changes in this motif. Cross-linking activity ofHomer proteins has demonstrated effects on glutamate receptor signalingand this action is due to the formation of signaling complexes that linkcell-surface receptors with intracellular receptors. Cross-linking byHomer proteins may also have consequences on receptor trafficking orother cellular functions of the interacting proteins.

Development of agents that modulate activity of the Homer EVH1 domain isfurthered by knowledge of the crystal structure of Homer protein. Themethod includes designing inhibitors of Homer protein that formnon-covalent bonds with amino acids in the Homer binding sites basedupon the crystal structure co-ordinates of Homer protein binding domain;synthesizing the inhibitor; and determining whether the inhibitorinhibits the activity of Homer protein.

The “Homer protein binding domain” is a conserved sequence of aminoacids in the amino-terminal region of the that interacts with otherproteins. All Homer proteins possess a conserved region of about 175amino acids at their amino-termini. The 110 terminal amino acids in thisregion interact with the carboxy-termini of other proteins, for examplemetabotropic glutamate receptors, inositol trisphosphate receptors,Shank, and the like. The carboxy-termini region of the proteins to whichthe Homer protein binding domain may bind usually contains an amino acidsequence that contains a high number of proline residues.

One aspect of the invention resides in the obtaining of crystals ofHomer protein of sufficient quality to determine the three dimensional(tertiary) structure of the protein by X-ray diffraction methods. Theknowledge obtained concerning Homer proteins may be used in thedetermination of the three dimensional structure of the binding domainof Homer proteins. The binding domain can also be predicted by variouscomputer models. Upon discovering the three-dimensional proteinstructure of the binding domain, small molecules which mimic thefunctional binding of Homer protein to its ligands can be designed andsynthesized This is the method of “rational” drug design. Anotherapproach to “rational” drug design is based on a lead compound that isdiscovered using high thoughput screens; the lead compound is furthermodified based on a crystal structure of the binding regions of themolecule in question. Accordingly, another aspect of the invention is toprovide material which is a starting material in the rational design ofdrugs which mimic or prevents the action of Homer proteins.

The term “crystal structure coordinates” refers to mathematicalcoordinates derived from mathematical equations related to the patternsobtained on diffraction of a monochromatic beam of X-rays by the atoms(scattering centers) of a Homer protein molecule in crystal form. Thediffraction data are used to calculate an electron density map of therepeating unit of the crystal. The electron density maps are used toestablish the positions of the individual atoms within the unit cell ofthe crystal. The crystal structure coordinates of the Homer proteinbinding domain are obtained from a Homer protein crystal havingorthorhombic space group symmetry P2,212, with a=33.79, b=51.40, andc=66.30 Angstroms. The coordinates of the Homer protein binding domaincan also be obtained by means of computational analysis.

The term “selenomethione substitution refers to the method of producinga chemically modified form of the crystal of Homer. The Homer protein isexpressed by bacterial in meida that is depleted in methionine andsupplement in selenomethionine. Selenium is thereby incorporated intothe crystal in place of methionine sulfurs. The location(s) of seleniumare determined by X-ray diffraction analysis of the crystal. Thisinformation is used to generate the phase information used to constructthree-dimensional structure of the protein.

The term “heavy atom derivatization” refers to the method of producing achemically modified form of the crystal of Homer. A crystal is soaked ina solution containing heavy metal atom salts or organometalliccompounds, which can diffuse through the crystal and bind to the surfaceof the protein. The location(s) of the bound heavy metal atom(s) aredetermined by X-ray diffraction analysis of the soaked crystal. Thisinformation is used to generate the phase information used to constructthree-dimensional structure of the protein.

Those of skill in the art understand that a set of structure coordinatesdetermined by X-ray crystallography is not without standard error.

The term “unit cell” refers to the basic parallelipiped shaped block.The entire volume of a crystal may be constructed by regular assembly ofsuch blocks.

The term “space group” refers to the arrangement of symmetry elements ofa crystal.

The term “molecular replacement” refers to a method that involvesgenerating a preliminary model of an Homer crystal whose structurecoordinates are not known, by orienting and positioning a molecule whosestructure coordinates are known. Phases are then calculated from thismodel and combined with observed amplitudes to give an approximateFourier synthesis of the structure whose coordinates are known.

The crystal structure coordinates of Homer protein may be used to designcompounds that bind to the protein and alter its physical orphysiological properties in a variety of ways. The structure coordinatesof the protein may also be used to computationally screen small moleculedata bases for compounds that bind to the protein. The structurecoordinates of Homer mutants (e.g., missense mutations, deletionmutations, and the like, obtained by site-directed mutagenesis, byexposure to mutagenic agents, through selection of naturally occurringmutants, etc.) may also facilitate the identification of relatedproteins, thereby further leading to novel therapeutic modes fortreating or preventing Homer-mediated conditions. A potential inhibitoris designed to form hydrogen bonds with tryptophan²⁴, phenylalanine⁷⁴,threonine⁶⁶, threonine⁶⁸, glutamine76, alanine⁷⁸, threonine⁷⁰, andvaline⁸⁵ of the Homer binding domain.

A method is also provided for identifying a compound that affects theformation of cell surface receptors into clusters. The method includesincubating the compound and a cell expressing a Homer protein and aHomer Interacting protein, such as a Shank protein, a Homer InteractingProtein, and the like, under conditions sufficient to allow the compoundto interact with the cell, determining the effect of the compound on theformation of cell-surface receptors into clusters, and comparing theformation of cell-surface receptors into clusters in cells contactedwith the compound with the formation of cell surface receptors intoclusters in cells not contacted with the compound.

Shank proteins are a novel family of proteins found at the postsynapticdensity (PSD) and which are capable of binding to other proteins. Shankproteins contain multiple protein interaction domains, including ankyrinrepeats, SH3 domain, PDZ domain, at least one proline rich domain and atleast one SAM domain. The PDZ domain of Shank mediates binding to thecarboxy-terminus of guanylate kinase associated protein (GKAP), and thisinteraction is important in neuronal cells for the synaptic localizationof Shank proteins. Shank proteins also interact with Homer proteins andtherefore Shank and Homer may serve as a protein bridge that linksspecific proteins that bind to Homer and specific proteins that bind toShank. Exemplary Shank proteins include Shank 1a, Shank 1b and Shank 3,and cortactin binding protein, and the like.

A compound can affect the formation of cell-surface receptors intoclusters by either stimulating the formation of cell-surface receptorsinto clusters or by inhibiting the recruitment of cell-surface receptorsinto clusters. When the effect is “inhibition”, cell-surface clusteringis decreased as compared with the level in the absence of the testcompound. When the effect is “stimulation”, cell-surface clustering isincreased as compared to a control in the absence of the test compound.

A method is further provided for treating a subject with a disorderassociated with metabotropic receptors or ion channel receptorscomprising administering to the subject a therapeutically effectiveamount of a compound that modulates Homer protein activity. In yetanother embodiment, a method is provided for treating a subject with adisorder associated with Homer protein activity, comprisingadministering to the subject a therapeutically effective amount of acompound that modulates Homer protein activity.

Essentially, any disorder that is etiologically linked to a glutamatereceptor, an inositol trisphosphate receptor, a ryanodine receptor, aShank protein, I42 (or other Homer interacting proteins) or to a Homerprotein could be considered susceptible to treatment with an agent thatmodulates Homer protein activity. The disorder may be a neuronal celldisorder. Examples of neuronal cell disorders include but are notlimited to Alzheimer's disease, Parkinson's disease, stroke, epilepsy,neurodegenerative disease, Huntington's disease, and brain or spinalcord injury/damage, including ischemic injury. The disorder may also bea disorder of a cardiac disorder, a disorder of musculature, a renaldisorder, a uterine disorder or a disorder of bronchial tissue. Thedisorder may be epilepsy, glutamate toxicity, a disorder of memory, adisorder of learning or a disorder of brain development.

Detection of altered (decreased or increased) levels of “Homer proteinactivity” can be accomplished by hybridization of nucleic acids isolatedfrom a cell of interest with a Homer polynucleotide of the invention.Analysis, such as Northern Blot analysis, are utilized to quantitateexpression of Homer, such as to measure Homer transcripts. Otherstandard nucleic acid detection techniques will be known to those ofskill in the art. Detection of altered levels of Homer can alsoaccomplished using assays designed to detect Homer polypeptide. Forexample, antibodies or petides that specifically bind a Homerpolypeptide can be utilized. Analyses, such as radioimmune assay orimmunohistochemistry, are then used to measure Homer, such as to measureprotein concentration qualitatively or quantitatively.

Treatment can include modulation of Homer activity by administration ofa therapeutically effective amount of a compound that modulates Homer orHomer protein activity. The term “modulate” envisions the suppression ofHomer activity or expression when Homer is overexpressed or has anincreased activity as compared to a control. The term “modulate” alsoincludes the augmentation of the expression of Homer when it isunderexpressed or has a decreased activity as compared to a control. Theterm “compound” as used herein describes any molecule, e.g., protein,nucleic acid, or pharmaceutical, with the capability of altering theexpression of Homer polynucleotide or activity of Homer polypeptide.Treatment may inhibit the interaction of the EVH1 domain of Homer withits target protein , may increase the avidity of this interaction bymeans of allosteric effects, may block the binding activity of thecoiled-coil doamin of Homer or influence other functional properties ofHomer proteins.

Candidate agents include nucleic acids encoding a Homer, or thatinterfere with expression of Homer, such as an antisense nucleic acid,ribozymes, and the like. Candidate agents also encompass numerouschemical classes wherein the agent modulates Homer expression oractivity.

Where a disorder is associated with the increased expression of Homer,nucleic acid sequences that interfere with the expression of Homer canbe used. In this manner, the coupling of cell-surface and intracellularreceptors can be inhibited. This approach also utilizes, for example,antisense nucleic acid, ribozymes, or triplex agents to blocktranscription or translation of Homer mRNA, either by masking that mRNAwith an antisense nucleic acid or triplex agent, or by cleaving it witha ribozyme in disorders associated with increased Homer. Alternatively,a dominant negative form of Homer polypeptide could be administered.

When Homer is overexpressed, candidate agents include antisense nucleicacid sequences. Antisense nucleic acids are DNA or RNA molecules thatare complementary to at least a portion of a specific mRNA molecule(Weintraub, 1990, Scientific American, 262:40). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetcell. The use of antisense methods to inhibit the in vitro translationof genes is well known in the art (Marcus-Sakura, 1988, Anal.Biochem.,172:289).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., 1991,Antisense Res. and Dev., 1(3):227; Helene, C., 1991, Anticancer DrugDesign, 6(6):569).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, 1988, J.Amer.Med. Assn., 260:3030). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, 1988, Nature, 334:585) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

When a disorder is associated with the decreased expression of Homer,nucleic acid sequences that encode Homer can be used. An agent whichmodulates Homer expression includes a polynucleotide encoding apolypeptide of SEQ ID NO:2, 4, 6, 8, 10 or 12, or a conservative variantthereof. Alternatively, an agent of use with the subject inventionincludes agents that increase the expression of a polynucleotideencoding Homer or an agent that increases the activity of Homerpolypeptide.

In another embodiment of the invention, there is provided a transgenicnon-human animal having a transgene that expresses Homer 1achromosomally integrated into the germ cells of the animal. Animals arereferred to as “transgenic” when such animal has had a heterologous DNAsequence, or one or more additional DNA sequences normally endogenous tothe animal (collectively referred to herein as “transgenes”)chromosomally integrated into the germ cells of the animal. Thetransgenic animal (including its progeny) will also have the transgenefortuitously integrated into the chromosomes of somatic cells.

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods may be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191. In yet another such method, embryonic cells areinfected with a retrovirus containing the transgene whereby the germcells of the embryo have the transgene chromosomally integrated therein.When the animals to be made transgenic are avian, because avianfertilized ova generally go through cell division for the first twenty hin the oviduct, microinjection into the pronucleus of the fertilized eggis problematic due to the inaccessibility of the pronucleus. Therefore,of the methods to make transgenic animals described generally above,retrovirus infection is preferred for avian species, for example asdescribed in U.S. Pat. No. 5,162,215. If microinjection is to be usedwith avian species, however, a recently published procedure by Love etal., (Biotechnology, Jan. 12, 1994) can be utilized whereby the embryois obtained from a sacrificed hen approximately two and one-half h afterthe laying of the previous laid egg, the transgene is microinjected intothe cytoplasm of the germinal disc and the embryo is cultured in a hostshell until maturity. When the animals to be made transgenic are bovineor porcine, microinjection can be hampered by the opacity of the ovathereby making the nuclei difficult to identify by traditionaldifferential interference-contrast microscopy. To overcome this problem,the ova can first be centrifuged to segregate the pronuclei for bettervisualization.

The “non-human animals” of the invention are murine typically (e.g.,mouse). The “transgenic non-human animals” of the invention are producedby introducing “transgenes” into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for microinjection. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host gene before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic i.e.,animals which include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

In the microinjection method useful in the practice of the subjectinvention, the transgene is digested and purified free from any vectorDNA e.g. by gel electrophoresis. It is preferred that the transgeneinclude an operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionin, skeletal actin,P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, andthymidine kinase. Promoters for viral long terminal repeats (LTRs) suchas Rous Sarcoma Virus can also be employed. Constructs useful in plasmidtransfection of embryonic stem cells will employ additional regulatoryelements well known in the art such as enhancer elements to stimulatetranscription, splice acceptors, termination and polyadenylationsignals, and ribosome binding sites to permit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 8:6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6:383-388, 1987). Alternatively, infection can be performedat a later stage. Virus or virus-producing cells can be injected intothe blastocoele (D. Jahner et al., Nature 2-98:623-628, 1982). Most ofthe founders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic nonhumananimal. Further, the founder may contain various retro viral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (D. Jahneret al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (M. J. Evans et al. Nature292:154-156, 1981; M. O. Bradley et al., Nature 309: 255-258, 1984;Gossler, et al., Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986; andRobertson et al., Nature 322:445-448, 1986). Transgenes can beefficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science 240:1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous nucleic acid molecule. “Heterologous” refers to a nucleicacid sequence that either originates from another species or is modifiedfrom either its original form or the form primarily expressed in thecell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode Homer protein-sense and antisensepolynucleotides, which may be expressed in a transgenic non-humananimal. The term “transgenic” as used herein additionally includes anyorganism whose genome has been altered by in vitro manipulation of theearly embryo or fertilized egg or by any transgenic technology to inducea specific gene knockout. As used herein, the term “transgenic” includesany transgenic technology familiar to those in the art which can producean organism carrying an introduced transgene or one in which anendogenous gene has been rendered non-functional or “knocked out”.

Antibodies of the invention may bind to Homer proteins or Homerinteracting proteins provided by the invention to prevent normalinteractions of the Homer proteins and Homer Interacting proteins.Binding of antibodies to Homer proteins or Homer Interacting Proteinscan interfere with cell-signaling by interfering with an intracellularsignaling pathway. Binding of antibodies can interfere with Homerprotein binding to extracellular receptors, e.g., to NMDA receptors, tometabotropic receptors, and the like. Binding of antibodies caninterfere with Homer protein binding to intracellular receptors, e.g.,inositol trisphosphate receptors, and the like. Furthermore, binding toHomer proteins or to Homer Interacting Proteins can interfere withcell-surface receptor clustering mediated by Homer family proteins.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding to an epitopic determinant present in an inventionpolypeptide. Such antibody fragments retain some ability to selectivelybind with its antigen or receptor.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975).

Antibodies which bind to an invention polypeptide of the invention canbe prepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. For example, it may bedesirable to produce antibodies that specifically bind to the N- orC-terminal domains of an invention polypeptide. The polypeptide orpeptide used to immunize an animal is derived from translated cDNA orchemically synthesized and can be conjugated to a carrier protein, ifdesired. Commonly used carrier proteins which may be chemically coupledto the immunizing peptide include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), tetanus toxoid, and the like.

Invention polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See, for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising thepolypeptides of the invention. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyi, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal and polyclonal antibodies of the invention forthe in vivo detection of antigen, e.g., Homer, the detectably labeledantibody is given a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledantibody is administered in sufficient quantity to enable detection ofthe site having the antigen comprising a polypeptide of the inventionfor which the antibodies are specific.

The concentration of detectably labeled antibody which is administeredshould be sufficient such that the binding to those cells having thepolypeptide is detectable compared to the background. Further, it isdesirable that the detectably labeled antibody be rapidly cleared fromthe circulatory system in order to give the best target-to-backgroundsignal ratio.

As a rule, the dosage of detectably labeled antibody for in vivotreatment or diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. Such dosages may vary, forexample, depending on whether multiple injections are given, antigenicburden, and other factors known to those of skill in the art.

The following examples are intended to illustrate but not to limit theinvention in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used.

EXAMPLES

Homer 1a is an IEG and is the original member of a family of proteinsthat function together as a regulated adapter system that ishypothesized to control the coupling of membrane receptors tointracellular pools of releasable calcium. Homer proteins function atexcitatory synapses to couple membrane group 1 metabotropic glutamatereceptors (mGluR) to endoplasmic reticulum-associated inositoltrisphosphate receptors (IP3R) (Brakeman et al., 1997; Tu et al., 1998;Xiao et al., 1998). Current studies suggest a broader role for Homerproteins in calcium signaling and receptor trafficking. The Shank familyof proteins was identified based on their association with Homer(Naisbitt et al., 1999; Tu et al., 1999). Shank, together with Homer,appears to be part of both the NMDA and group 1 mGluR signalingcomplexes. By virtue of its interaction with Shank, Homer provides amechanism to couple NMDA Ca2+ influx to intracellular Ca2+-induced Ca2+release pools. The inventors have identified additionalHomer-interacting proteins that provide insight into the role of Homerin trafficking of group 1 mGluR (e.g., SEQ ID NOS:16, 18, 20). Becausethese Homer-dependent cellular processes are regulated by the IEG formof Homer (Homer 1a), mechanisms by which Homer proteins can modulateCa2+ dynamics of mGluR and NMDA receptors, as well as regulate receptortrafficking are defined.

Homer family proteins possess an N-terminal EVH1 domain that mediatesinteractions with mGluRs, IP3R, Shank and other novel proteins. The EVH1domain has been determined to bind the proline rich motif PPXXFR (Tu etal., 1998). The present invention provides the crystal structure of theHomer EVH1 domain. In complementary studies, genetic approaches wereused to identify critical residues in both the EVH1 domain and theligand that modulate the affinity of the Homer-mGluR (and otherHomer-interacting proteins) interaction. This information is essentialto an understanding of the integrative cellular actions of Homerproteins,. Together, these studies define the molecular basis ofspecificity of EVH1 interaction with its ligands, and provide insightinto how the EVH1 interaction is regulated,

This patent application includes a description of severalHomer-interacting proteins that are part of the signaling network thatis controlled by Homer (e.g., SEQ ID NOS: 16, 18, 20). Yeast two-hybridscreens and searches of NCBI protein data bases identified a set ofknown and novel candidate interacting proteins for Homer include theryanodine receptor, NMDA receptor subunit NR2D, human InaD and novelinteracting proteins termed I42 and I30. As described below, currentdata indicate that agents can be developed that specifically modulatethe crosslinking activity of Homer for these various receptors andthereby provide novel theraputics that regulate the output of thesereceptors on cellular function.

Homer acts in several ways to regulate cellular function. Homer andHomer-related proteins function as an adapter system to couple membranereceptors to intracellular pools of releasable Ca. This “signaling”function of Homer is documented in Xiao et (1998), Tu et al (1998 and1999) Naisbett et al. (1999), as well as by studies of the novelHomer-interacting protein termed 142 (see below). By virtue of itscrosslinking activity, Homer proteins play a role in synaptogenesis andspatial targeting/trafficking of GluRs to other postsynaptic structuralproteins. This function of Homer is supported by observations in Tu etal (1999) and Naisbett et al (1999).

Initial Cloning of Homer; a Novel Brain Immediate Early Gene (IEG)

Homer was cloned in a differential screen of seizure-stimulatedhippocampus. Prior work, in which IEG induction was examined in brainprovided a detailed understanding of time course and tissue distributionof the IEG response (Cole et al., 1989; Saffen et al., 1988; Worley etal., 1990), and suggested a paradigm to maximally induce novel IEG mRNAs(Lanahan and Worley, 1998; Worley et al., 1990). Once cloned, in situhybridization was used to screen for IEGs that were regulated in otherparadigms that activate neurons including LTP stimulation in thehippocampus (Brakeman et al., 1997) and acute administration of cocaine.In these models, Homer was one of the most highly induced of all of theIEGs (Brakeman et al., 1997). Initial characterization of Homer waschallenging in that the mRNA was nearly 7 kb, while the best deducedopen reading frame was only 186 aa, and was located near the 5′ end ofthe cDNA (Brakeman et al., 1997). The 3′ UTR was over 5 kb. The ORF wasconfirmed by in vitro transcription and translation of the cDNA, andrabbit polyclonal antisera were generated against bacterially expressedfusion proteins. With these antibodies, we were able to demonstrate thatthe protein was rapidly and transiently induced in the hippocampusfollowing a seizure (Brakeman et al., 1997). This confirmed the deducedORF and assured us that the cDNA was indeed translated in brain.

Homer Selectively Binds Group 1 Metabotropic Receptors and is Enrichedat Synapses

In an effort to discover the function of Homer, a yeast 2-hybridtechnique (Chevray and Nathans, 1992; Fields and Song, 1989) was used toscreen a cDNA library prepared from rat hippocampus and cortex. The fulllength Homer IEG was used as bait. Among ˜30 confirmed interactingcDNAs, one encoded the C-terminal 250 aa of mGluR5. We initiallyconfirmed that the proteins bind using a GSTHomer in a pulldown assaywith either fragments of mGluR5, or full length mGluR5 expressed inheterologous cells (HEK293 cells) (Brakeman et al., 1997). Homer proteinalso bound to mGluR1a, but not mGluR2, 3, 4, or 7. This was aninteresting clue to the function of Homer since mGluR1 and mGluR5(termed group 1 metabotropic receptors) couple to phospholipase C andactive hydrolysis of phosphoinositides to generate inositoltrisphosphate and diacylglycerol (Nakanishi et al., 1994). mGluR1a and 5also share sequence similarity in their long, cytosolically disposedC-terminus. Other metabotropic glutamate receptors (termed group 2 and3) inhibit adenylate cyclase activity, and have short C-termini thatlack homology to group 1 receptors. We proceeded to test whether Homerand mGluR5 naturally associate in brain and confirmed that theseproteins co-immunoprecipitate from detergent extracts of hippocampus(Brakeman et al., 1997). The next major clue was provided by theobservation that Homer immunoreactivity was enriched at excitatorysynapses (Brakeman et al., 1997). In brain, Homer protein was associatedwith dendrites and showed a punctate pattern consistent with alocalization in spines. The binding properties and cellular distributionof Homer suggested a role at the excitatory synapse.

Homer is a Member of a Family of Closely Related Proteins that areEnriched at the Excitatory Synapse

A search of the NCBI sequence data base identified several ESTs thatshowed strong homology to Homer, but were clearly distinct in that theyencoded additional C-terminal sequence (Brakeman et al., 1997). Using acombination of screening strategies, a family of 12 cDNAs was identifiedfrom rat, mouse, Drosophila, and human (Xiao et al., 1998). All of thesecDNAs encoded proteins with a similar protein structure and were deducedto be the products of 3 independent mammalian genes (termed Homer 1, 2,3) and 1 Drosophilia gene. Like Homer IEG (now termed Homer 1a), all newfamily members contain an N-terminal, ˜110 amino acid domain that bindsmGluR1a/5 ((Xiao et al., 1998). The region of Homer that interacts withmGluR1a/5 is termed an EVH1 domain based on its modest homology (20-25%identity) to domains in a family of proteins that include DrosophilaEnabled Gertler, 1996, mammalian VASP (Haffner et al., 1995) and theWiscott-Aldridge protein (WASP) (Ponting and Phillips, 1997; Symons etal., 1996). The EVH1 domains of Homer proteins from Drosophilia, rodentand human are conserved at a level of 80% identity (Xiao et al., 1998).Other than the IEG Homer 1a, all new forms of Homer encode an additionalC-terminal domain with predicted coiled-coil (CC) structure.

As the nomenclature suggests, Homer 1 gene encodes both the IEG form(Homer 1a) and splice forms that encode CC domains termed Homer 1b and1c. The 1b and 1c splice forms differ in their inclusion of anapproximately 10 amino acid sequence located between the EVH1 and CCdomains. (Homer family members that encode CC domains are also referredto as CC-Homers to distinguish them from Homer 1a, which lacks a CCdomain.) Similarly, Homer 2 encodes two CC-Homer splice forms termedHomer 2a and 2b, which also differ by a short internal sequence betweenEVH1 and CC domains. Homer 3 encodes a single form. The CC domains areless conserved than the EVH1 domain (˜40% identity between rat Homer 1,2 and 3) but they are able to specifically bind to themselves and toCC-domains of other Homer family members (Xiao et al., 1998). Homer CCdomains do not interact with other representative CC-domain proteins inGST pulldown assays, and a yeast 2-hybrid screen of brain cDNA with theCC-domain of Homer 1 identified multiple copies of Homer 1, Homer 2 andHomer 3, but not other CC domains (Xiao et al., 1998). As evidence thatHomer proteins can naturally self-multimerize, we demonstrated thatHomer 1b/Homer 3 heteromultimers co-immunoprecipitate from brain (Xiaoet al., 1998). These observations indicate that the Homer CC domainsmediate specific self-association.

In contrast to Homer 1a, all CC-containing Homer family members areconstitutively expressed in brain (Xiao et al., 1998). This wasconfirmed using both Northern blot and in situ hybridization assayswhich compared expression with Homer 1a in the same material. mRNA andprotein expression of Homer 1b/c, Homer 2 and Homer 3 are unchanged inhippocampus following a seizure while Homer 1a mRNA and protein areinduced at least 10 fold.

Antibodies were generated that specifically recognize each of theCC-Homers. Antibodies were raised against synthetic C-terminal peptidesequences. Because Homer 1b and 1c possess identical C-termini, theC-terminal antibodies recognize both splice forms. Similarly, C-terminalHomer 2 antibodies recognize both Homer 2a and 2b. Accordingly, whenusing these antibodies to detect Homer proteins, we refer to theimmunoreactivity as Homer 1b/c or Homer 2a/b. We used these antibodiesto determine that Homer 1b/c and 3 are enriched in a detergent resistantfraction of the postsynaptic density (PSD) (Xiao et al., 1998). Homer2a/b is also enriched in synaptic fractions, but is relatively moresoluble than Homer 1b/c and Homer 3. Like Homer 1a, each of theCC-Homers co-immunoprecipitates with group 1 mGluRs from brain (Xiao etal., 1998). Immunogold electron microscopy (EM) demonstrated that Homer1b/c and Homer 3 are ultrastructurally localized at the PSD (Xiao etal., 1998). These observations suggest that CC-Homer proteins functionas multivalent adapter complexes that bind mGluRs at postsynaptic sites.

Homer 1a Functions as a Natural Dominant Negative Protein

The fact that Homer 1a lacks a CC domain suggested that it may functionas a natural dominant negative to disrupt cross-linking of CC-Homers. Inthis model, the EVH1 domain of Homer 1a can bind and compete for thesame target proteins as CC-Homers (such as mGluR5), but because Homer 1alacks the CC-domain, it cannot self-associate and cannot cross-link. Totest the dominant negative hypothesis, we generated a transgenic mousethat constitutively expressed Homer 1a in brain neurons under thecontrol of a modified Thy-1 promoter Aigner, 1995 #200. We confirmedtransgene expression in hippocampus, cerebellum and cortex in twoindependent lines (Xiao et al., 1998). The level of transgene expressionin the hippocampus was similar to natural Homer 1a expression induced bya seizure. In contract to the natural Homer 1a, however, the transgenewas constitutively expressed in the unstimulated mouse. A prediction ofthe dominant negative hypothesis is that the ability toco-immunoprecipitate mGluR with Homer 1b/c or Homer 3 antibodies shouldbe diminished in the transgenic mouse. As one of the controls for thisexperiment, we demonstrated by western blot that levels of expression ofmGluR1a, mGluR5 and Homer 1b/c, 2a/b, 3 were unchanged in the transgenicmouse brain. We then performed IP experiments and observed theanticipated result; the co-immunoprecipitation of mGluR5 with CC-Homersfrom hippocampus was reduced in the transgenic mouse (Xiao et al.,1998). Similar co-immunoprecipitations of mGluR1a with Homer 3 fromcerebellum was also reduced. As an additional control, we demonstratedthat the ability to co-immunoprecipitate Homer 1b/c with Homer 3 was notaltered in the transgenic mouse. This was the predicted result since theassociation between these proteins is mediated by their CC domains, andthis interaction is not altered by the Homer 1a EVH1 domain. Theseobservations support the hypothesis that Homer 1a functions as a naturaldominant negative to regulate CC-Homer-dependent cross-linking.

Homer Binds a Proline Rich Sequence that is ˜50 aa from the C-terminusof Group 1 mGluRs

When we initially characterized the interaction between Homer andmGluR5, we anticipated that Homer might bind the free C-terminus. Thissurmise was based on the precedent of PDZ proteins such as PSD95 andGRIP, which bind the free C-terminus of NMDAR2 (Kornau, 1995) and AMPAreceptors (Dong et al., 1997). Homer was noted to encode a GLGF sequencelike the PDZ domain. Additionally, in GST pulldown assays that usedbrief washes, we noted a modest reduction of binding when the C-terminal4 or 10 aa were deleted from mGluR5 Brakeman, 1997 #99. (In retrospect,this modest reduction of binding may be due to Homer pulldown of Shankwhich does bind the free C-terminus of mGluR5, but appears to be loweraffinity than Homer-mGluR5 binding; see below.) However, with morestandard wash conditions, it became clear that the 4 and 10 aaC-terminal deletion mutants of mGluR5 continued to bind avidly to Homer.We continued the deletion strategy until we found that a 50 aaC-terminal deletion of mGluR5 destroyed binding to Homer. By contrast, a41 aa deletion of mGluR5 retained full binding activity. We noted thatthe intervening sequence was proline rich and shared sequence similaritywith the previously described SH3 ligand sequence Yu, 1994 #166 Weprepared a series of point mutants based on the known structure-functionrelationship for SH3 ligands. Binding assays confirmed generalcharacteristics of SH3 ligand binding, but also demonstrated that thatthe Homer binding site is distinct in the positioning of critical aminoacids (Tu et al., 1998). A consensus for binding was determined to bePPXXFR, consistent with the observation that mutation of either of theprolines or the phenylalanine, or a change in their relative position,interrupted binding. The arginine in the last position is preferred overother amino acids, but is not essential. Mutations were identicallyeffective in interrupting binding to each of the Homer family membersincluding Homer 1a, 1b/c, 2a/b, 3 and an EVH1 only fragment (110aa) ofHomer 1. Thus, we conclude that the interaction with mGluR5 is mediatedby the Homer EVH1 domain.

Mutations of mGluR5 were initially tested in the context of a 250aaC-terminal fragment, but were also determined to have an identicaleffect on binding when placed in the full length mGluR5 protein (Tu etal., 1998). This exquisite sensitivity of Homer binding to changes insingle amino acid within the Homer-ligand site has been confirmed inother Homer-interacting proteins including mGluR1a (Tu et al., 1998),Shank (Tu et al., 1999) and I42 (see below). To further confirm that theinteraction was mediated by a direct interaction at the Homer-ligandsite (as opposed to a secondary allosteric effect on a remote bindingsite), we prepared synthetic 10 mer peptides with either the wild type,or F-to-R mutation, and demonstrated that the wild type peptide blockedbinding of mGluR1a or mGluR5 to each of the Homer family members (Tu etal., 1998). Approximately half of the binding was blocked at a peptideconcentration of 3.4 micromolar. By contrast, the F-to-R mutant peptidedid not alter binding at concentrations as high as 340 micromolar.

Homer Binds the IP3 Receptor

Armed with a consensus sequence that predicted binding to Homer, wesearched the NCBI data base for other proteins that might bind Homer. AHomer-ligand site was identified in the IP3R, dynamin III, a human alphaadrenergic receptor and the ryanodine receptor (Tu et al., 1998). Eachof these interactions were determined to be consistent with the knowntopology of the candidate interacting protein, assuming that Homerproteins are cytosolic. We were able to confirm a biochemicalinteraction of Homer with the IP3R and dynamin III using GST pull downassays. More importantly, we demonstrated that the IP3Rco-immunoprecipitates with each of the Homer 1b/c, 2a/b and 3 fromdetergent extracts of cerebellum (Tu et al., 1998). Homer appears to beassociated with a substantial portion of IP3R in the cerebellum, since acocktail of the three Homer antibodies is able to specifically (comparedto a cocktail of preimmune serums) co-immunoprecipitate ˜50% of thetotal IP3R in detergent extracts (CHAPS).

CC-Homers Function to Link mGluR5 and IP3R in a Signaling Complex

Based on the prior observations, we examined the hypothesis thatCC-Homers might cross-link mGluR and IP3R. This notion was appealing inthat the IP3R is part of the signaling network that is activated uponglutamate stimulation of mGluR1/5. Signaling complexes had previouslybeen described including; AKAP proteins which function as scaffolds forspecific kinases and their substrates Lester, 1997 #149, and theDrosophila protein InaD which couples the membrane light activatedchannel with its down stream effector enzyme, phospholipase C Tsunoda,1997 #147. Unlike these other examples of signaling complexes, however,Homer would need to form a bridge between receptors in two differentmembranes. Functional mGluRs are in the plasma membrane while the IP3Ris localized primarily to intracellular endoplasmic reticulum (ER). Insupport of the notion that ER and plasma membranes can come in closeapposition in neurons, we noted that Dr. Kristin Harris (Harvard)described the presence of smooth ER (SER, or spine apparatus) in thespines of hippocampal and cerebellar neurons (Tu et al., 1998).Remarkably, the SER forms close appositions with the plasma membranethat were uniquely localized to the lateral margin of the PSD. Thesesites are precisely where the group 1 mGluRs are localized (Baude etal., 1993; Lujan et al., 1997; Nusser et al., 1994). The IP3R is presentin spines of cerebellar Purkinje neurons where it is associated with thespine apparatus (Satoh et al., 1990). (Interestingly, in hippocampalneurons, the RYR is present in the spine apparatus while the IP3Rappears to be restricted to the dendritic shaft reviewed in (Narasimhanet al., 1998). Homer 1b/c and 3 are also enriched in the cytosol at thelateral margin of the PSD (Xiao et al., 1998). Thus, available anatomicevidence supported the notion that synaptic mGluRs come in closeapposition with SER-associated IP3Rs at sites that are enriched forCC-Homers.

As a first test of the hypothesis the CC-Homers cross-link mGluR andIP3R, we asked whether we could detect a trimolecular complex of mGluR,Homer and IP3R in brain. Indeed, IP3R antibody specificallyco-immunoprecipitated Homer and mGluR1a from cerebellum (Narasimhan etal., 1998). Since IP3Rs are not known to directly interact with mGluR1a,this result supported the hypothesis that Homer bridges these proteinsto form a trimolecular signaling complex. A further prediction of the“Homer hypothesis” is that Homer 1a should uncouple the putativemGluR-CC-Homer-IP3R complex. To test this, we monitored the effect ofHomer 1a expression on glutamate-induced intracellular calcium release.Plasmids expressing Homer 1a or Homer 1b were transfected along withgreen fluorescent protein (gene gun) and identified Purkinje neuronswere stimulated with quisqualate. A patch electrode containing the Ca2+detector Fura-2 was attached to the soma and a holding potential of −60mV was applied. Tetrodotoxin and picrotoxin were included in the bath toblock synaptic input and EDTA/MgCl₂ was included to assure that measuredCa2+ increases in the cell were generated from intracellular stores.Under these conditions, quiqualate-induced Ca2+ increases are due tomGluR1-evoked release from IP3R pools (Roche et al., J Biol Chem (1999)274:25953-259577). Expression of Homer 1b did not alter the induced Ca2+transient compared to cells transfected with an empty vector. Bycontrast, neurons transfected with Homer 1a showed a Ca2+ transient thatwas reduced in amplitude and delayed in time to peak (Tu et al., 1998).This result is consistent with the notion that the IP3 generated bymGluR1a activation of phospholipase C is less effective in releasingCa2+ from the Ip3R pools in neurons expressing Homer 1a, and isanticipated if Homer 1a disrupts the physical linkage between mGluR1aand IP3R. Released IP3 must diffuse further, thereby resulting in alower effective concentration of IP3 at the receptor.

CC-Homers Alter Trafficking of mGluR1a/5 in Heterologous Cells

We initiated studies to examine the effect of Homer on mGluR5expression. When wild type mGluR5 was expressed in heterologous cells(HEK293, COS or HeLa) the receptor reached the plasma membrane surfacewhere it was diffusely localized. This was also true when mGluR5 wasco-expressed with Homer 1a. However, we noted that co-expression ofmGluR5 with Homer 1b resulted in intracellular inclusions of mGluR5(Roche et al., 1999 supra). This effect of Homer 1b was dependent on theamount of transfected plasmid and was most obvious when equal amounts ofHomer 1b and mGluR5 plasmids were co-transfected. There was a trend forhigher level expression of mGluR5 when co-transfected with Homer 1b.When ratios of transfected plasmids were titrated so that total mGluR5expression was the same (comparing expression with or without Homer 1b),a substantial portion of the total mGluR5 was associated with theintracellular pool when co-expressed with Homer 1b. In these cells,relatively less reached the plasma membrane compared to mGluR expressedalone, or co-expressed with Homer 1a. We further noted that at earliertimes after transfection of Homer 1b and mGluR5, mGluR5 showed anenrichment in perinuclear organelles with a reticular pattern throughoutthe cell that resembled the ER. To assess the nature of theCC-Homer-dependent cellular accumulation, we compared the distributionof mGluR5 with the ER specific maker BIP B (Roche et al., 1999, supra).Staining with BIP antibodies revealed extensive ER present in bothtransfected and untransfected cells and co-localization with mGluR5. Wealso noted that the perinuclear organelles were not present withinnon-transfected cells and therefore appeared to be ER-derived structuresunique to cells overexpressing mGluR5 and Homer 1b. These observationssuggest that Homer 1b, but not Homer 1a, causes mGluR5 to be retained inthe ER.

As an additional assay for ER retention, we examined the status of thecarbohydrates present on mGluR5 in cells co-expressing Homer 1a or Homer1b. If Homer 1b caused mGluR5 to be retained within the ER, then mGluR5should contain immature, high mannose carbohydrates which are sensitiveto digestion with the enzyme Endoglycosidase H (Endo H). Alternatively,if mGluR5 had successfully traveled through the ER and cis Golgi, itwould possess mature, complex carbohydrates which would be Endo Hresistant. Mature carbohydrates would be anticipated if mGluR5 was onthe cell surface or if it was sequestered in a post-Golgi intracellularcompartment such as endosomes. We determined that mGluR5 is Endo Hresistant when expressed alone or with Homer 1a (Xiao et al., 1998).However, when expressed with H1b, mGluR5 is Endo H sensitive, consistentwith the hypothesis that expression of H1b leads to the retention ofgroup I mGluR in the ER.

The subcellular localization of the group II metabotropic glutamatereceptor mGluR2 was the same whether expressed alone or with H1b. Inaddition, we used a series of mGluR5 constructs containing pointmutations within the Homer binding site and found that mutations thatdisrupt mGluR5/Homer interactions in vitro also prevented ER retentionof mGluR5 co-expressed with H1b (Takei et al., 1994). mGluR5 P1125L,which does not bind to Homer in vitro (Tu et al., 1998), was notretained in the ER when co-expressed with H1b. In contrast, mGluR5S1126F, which does bind Homer in vitro, was ER retained whenco-expressed with H1b. Other point mutations in adjacent residues wereanalyzed and the results were consistent with in vitro binding studiessummarized in B (Ikeda et al., 1995), demonstrating that mGluR5 isretained within the ER by H1b only when its Homer binding site isintact.

While these experiments were performed in heterologous cells, we alsonoted enrichment of the group I metabotropic receptor mGluR1a in the ERof Purkinje cells (Kammermeier et al., submitted). Since Purkinjeneurons express particularly high levels of CC-Homers (Xiao et al.,1998), this suggests Homer proteins may naturally regulate receptortrafficking through the ER. In this model, Homer 1a would be permissivefor transfer through the ER Golgi system to insertion into thepostsynaptic membrane. The ability of CC-Homers to alter the spatialdistribution and metabolism of ER associated proteins may also impactthe IP3R. IP3Rs in Purkinje neurons are associated with dense stacks ofER (Satoh et al., 1990) and this stacking morphology has been shown tobe regulated by neural activity (Takei et al., 1994). Since asubstantial portion of IP3R in cerebellum is associated with CC-Homers,it is possible that the ability of CC-Homer to crosslink interactingproteins on two adjacent membranes plays a regulatory role in ERmorphology and function. Experiments in Aims 2 and 3 will examine thishypothesis.

Homer Modulates mGluR Coupling to Ion Channels

Group 1 mGluRs modulate ionic currents by activating pertussistoxin-sensitive and -insensitive G proteins (Naisbitt et al., 1999).Modulation of Ca2+ currents by heterologously expressed group 1 mGluRsin superior cervical ganglion (SCG) neurons proceeds through multiplepathways involving both the a and βg -subunits of G proteins. Weexamined the effect of Homer on mGluR coupling to Ca2+ and M-typepotassium channels in SCG neurons. CC-Homers, including 1b, 2b and 3produced a similar reduction of the effect of group 1 mGluRs (Kim etal., 1997; Naisbitt et al., 1999; Naisbitt et al., 1997; Takeuchi etal., 1997). By contrast, Homer Ia or an engineered short form of Homer 2did not block group 1 mGluR effects, but were able to partially reversethe effect of the CC-Homers.

Homer Interacts with Shank Suggesting a Role Synaptogenesis and NMDARFunction

To gain further insight into the physiological function of Homer, wecharacterized a novel family of proteins that were identified based ontheir interaction with Homer 1a in a yeast 2-hybrid screen of a braincDNA library. These Homer-interacting proteins were determined to beidentical to the Shank family of PSD proteins that interact with GKAPand the PSD-95 complex (Tu et al., 1999). Shank proteins arespecifically enriched at excitatory synapses and co-localize with NMDAreceptors in primary neuronal cultures (Naisbitt et al., 1999). Shankproteins appear to be recruited to excitatory synapses by virtue oftheir interaction with GKAP, a synaptic protein that binds to theguanylate kinase domain of PSD-95 (Kim et al., 1997; Naisbitt et al.,1999; Naisbitt et al., 1997; Takeuchi et al., 1997). In addition to thePDZ domain which binds GKAP, Shank contains domains that mediateself-multimerization and interaction with cortactin (Golshani et al.,1998). Shank also directly interacts with Homer (Lujan et al., 1997).Homer and Shank proteins co-localize at the PSD of CA1 pyramidal neurons(Tu et al., 1999), and native Homer-Shank complexes were identified inbrain using GST pull down assays of Shank with GKAP (Otani and Connor,1998). Additionally, Homer and Shank co-immunoprecipitate from brain(Aniksztejn et al., 1991; Ben-Ari et al., 1992). These observationsindicate that Shank and Homer naturally associate in brain. Biochemicalstudies indicate that the Shank-Homer interaction is mediated by theEVH1 domain of Homer which binds to a single Homer-ligand site presentin the proline-rich domain of Shank proteins (Tu et al., 1999). Aquaternary complex of Homer/Shank/GKAP/PSD-95 is assembled inheterologous cells, with Homer and PSD-95 co-localizing in largeclusters (Berridge, 1998). Thus, Shank provides a molecular bridge thatlinks the NMDA receptor complex with Homer and its associated proteins.

The Homer-Shank interaction also produces clustering of group 1 mGluRs(Satoh et al., 1990; Villa et al., 1992). Clustering molecules havepreviously been identified for a variety of receptors and ion channels(Selig et al., 1995), but Shank-Homer are the first clustering proteinsfor group 1 mGluR. It is notable that the mechanism of clusteringinvolves a linkage of mGluRs with the previously defined NMDA receptorscaffold. Thus the Shank-Homer interaction could be relevant tosynaptogenesis, by docking mGluRs to a preestablished “core” of NMDAreceptors. In support of such a mechanism, functional NMDA receptorsappear to precede the emergence of metabotropic receptors in thehippocampus and cerebellum (Xiao et al., 1998). Homer proteins, inassociation with Shank, could function to localize and cluster themGluRs in proximity to NMDARs, and may contribute to the perisynapticlocalization of group 1 metabotropic receptors (Lujan et al., 1997).

By linking NMDA and mGluR signaling pathways, the Shank-Homerinteraction might also contribute to examples of glutamate receptorcrosstalk for which physical proximity of molecules may be important,such as activation of phospholipase C (Beneken et al., submitted) orprotein kinase C (Aniksztejn et al., 1991; Ben-Ari et al., 1992).Additionally, the Homer/Shank/GKAP/PSD-95 assembly may mediate physicalassociation (and perhaps functional coupling) of the NMDAR with IP3R/RYRand intracellular Ca2+ stores. Consistent with such a functionalinteraction, recent studies indicate that NMDA receptor-dependentincreases in spine Ca2+ may derive from intracellular stores by amechanism of Ca2+-induced Ca2+ release (CICR) (Emptage et al., 1999) andreviewed by (Svoboda and Mainen, 1999). Both IP3R and ryanodine receptorchannels possess CICR properties (Berridge, 1998), and are similarlylocalized in dendrites and spines of specific neuronal types (Satoh etal., 1990; Villa et al., 1992). The physical proximity of glutamatereceptors with calcium pools may underlie synergistic effects of mGluRson NMDA-dependent responses as reported in studies of LTP (Bashir etal., 1993; Bortolotto et al., 1994) but see also ((Selig et al., 1995),and is consistent with the reduction of LTP in group 1 mGluR mutant mice(Prehoda et al., 1999) but see also (Conquet et al., 1994).

The proposed model for Shank and Homer-dependent clustering requiresthat Homer be multivalent in order to cross-link Shank/GKAP/PSD95 toIP3R/RYRs and to mGluRs. This is achieved by multimerization ofconstitutively expressed CC-Homers (Xiao et al., 1998). In this context,the monovalent Homer 1a IEG product appears to function to uncoupleproteins that are linked via the constitutively expressed CC-Homermultimers, and thereby dynamically regulate the assembly of thispostsynaptic network. Cocaine-induced increases in Homer 1a may thusmodulate both mGluR and NMDA Ca2+ responses in spines.

Homer EVH1 Domain Crystal Structure

To investigate the structural basis of interactions between EVH1 domainsand ligands, we determined the high-resolution crystal structure of theEVH1 domain from rat Homer 1 ( ). Methods of protein purification andcrystallization are described in our manuscript (Niebuhr et al., 1997;Tu et al., 1998). This structure revealed that the EVH1 module ishomologous to both the plextrin homology (PH) domain and thephosphotyrosine binding (PTB) domain. (legend next page)

At the same time we were working to solve the structure of Homer 1 EVH1,Dr. Wendel Lim's group (at UCSF) solved the structure of the relatedEVH1 protein termed Mena (20% identical to Homer EVH1 domain) (Prehodaet al., 1999). Comparison of the Mena and Homer coordinates confirmedthat these are related proteins despite the low degree of amino acididentity. The Mena crystal was solved with a 6mer peptide and identifieda putative ligand binding surface. Both of our groups determined thatco-crystals were not formed with longer synthetic peptides. One issuethat concerned us regarding the putative ligand-binding site on Mena wasthat the affinity of the 6mer used for Mena was 100 fold less than thatof a 10 mer (Prehoda et al., 1999). The measured affinity of the 6merwas ˜600 micromolar. Additionally, within the EVH1 family, Homer is oneof the most divergent members (Prehoda et al., 1999). One importantdifference between Mena and Homer EVH1 binding, is the orientation ofthe phenylalanine relative to the polyprolines. The optimal ligand forMena is FPPPP while the consensus ligand for Homer is PPXXF. This may beimportant since the F is the single most critical side chain for theinteraction when tested with larger peptides for both EVH1 domains(Niebuhr et al., 1997; Tu et al., 1998). In the Mena structure, the Fside chain is not placed in a clear hydrophobic pocket (the ring appearsto coordinate an arginine) and superposition of the ligand coordinatesin Homer EVH1 is even less obviously stabilized.

To examine the predictive power of the Mena co-crystal for the ligandbinding activity of Homer EVH1, we tested a series of missense mutationsthat targeted sites anticipated to contact the prolines of the ligand(PPXXF) sequence. Based on the homology of the EVH1 domain with the PTBdomain, we also tested sites on Homer that would be critical if Homermimicked the peptide binding surface of the PTB domain. This PTB ligandsite is remote from the putative Mena EVH1 ligand site. Our mutationanalysis also tested a series of mutants selected based on the homologybetween Homer and WASP. Genetic data from patients with Wiscott Aldrichsyndrome defined a series of mutations in the EVH1 domain that map tosites that are distinct from both the PTB and the putative Mena ligandsites. Our selection of the mutational substitutions was based on theHomer EVH1 structure. Substituted amino acids were selected to besufficiently conservative as not to disrupt the primary structure.

A total of 30 missense mutants of the Homer EVH1 domain were expressedin HEK293 cells and assayed for binding to either mGluR1a or Shank3using GST pulldown assays. Surface-exposed mutations within the regionhomologous to the peptide binding site of PTB domains had no affect onpeptide binding. Similarly, mutations based on the WASP data were alsoineffective in disrupting binding. By contrast, certain of the mutantsbased the Mena ligand site did disrupt Homer EVH1 binding. Despiteambiguities involved with interpreting the effects of any singlemutation, the nature and distribution of the effects of site-directedmutations in the Homer EVH1 domain on Homer-ligand interactions stronglyimplicate the Mena ligand region as mediating natural ligand binding bythe Homer EVH1 domain.

One interesting finding from our analysis of mutant Homer EVH1 bindingis that certain mutations disrupt binding specifically to mGluR1a, butnot to Shank3 (and visa versa). One interpretation of this finding isthat there are determinants of binding in addition to the core PPXXFmotif. An important implication of this observation is that differencesin critical determinants of Homer binding to its various targets may beexploited to develop pharmaceuticals that can selectively disruptinteractions with a particular target.

I42 Interacts with Homer

I42 (SEQ ID NOS:17 and 18) encodes a novel protein that was firstidentified in a Y2H screen of brain cDNA with the Homer EVH1 domain.Current information indicates that I42 functions with Homer at theexcitatory synapse. We have generated I42 specific antisera and candemonstrate robust co-immunoprecipitation of I42 with Homer from brain.ImmunoEM analysis demonstrates that I42 is localized to the postsynapticdensity. The predicted domain structure of I42 indicates that it sharescertain properties with Shank including a N-terminal structural domain(a band 4.1 domain in I42), a single PDZ domain, and a central prolinerich domain with a single Homer-ligand site. Additionally, there is aC-terminal type 1 PDZ ligand motif. We have identified a relatedsequence in the data base (KIAA sequence has several errors with frameshifts) suggesting that I42 may represent a gene family.

Current studies indicate a functional interaction of I42, Homer andmGluRs. We have performed a yeast 2-hybrid screen of the I42 PDZ domainand find it binds βB-Pix (also termed Cool-1) (Allen et al., 1998).β-Pix is a guanine nucleotide exchange factor (GEF) for Rac1/CDC42. Thisinteraction appears robust using GST pulldown assays and we haverecently confirmed the interaction using co-immunoprecipitation assaysfrom brain. Biochemical assays indicate that the PDZ domain of I42 bindsits own C-terminus (may be intra or inter molecular). Based on theseobservations, I42 functions as a scaffold/cytoskeletal regulatoryprotein that responds to specific signals and may link between mGluRactivation and Rac-dependent cytoskeletal remodeling. This biochemicalassociation may play a role in mGluR trafficking or synaptic remodeling.An additional functional consequence of the Homer I42 interaction isindicated by the demonstrated association of β-Pix with p21 activatedkinase (Pak) (Tu et al., 1999). Paks are a family of kinase that cansignal both locally and more distally to the nucleus. A mutation of Pak3has recently been linked to mental retardation (Tu et al., 1998),confirming the importance of this regulated kinase to cognitivefunction. Accordingly, I42 appears to be part of a novel signalingpathway for the mGluRs that may be regulated by Homer proteins.

In preliminary studies, we observe that I42 co-immunoprecipitates withHomer from brain. Antibodies for I42 also co-immunoprecipitates mGluR1from brain. In parallel studies, we observed the interaction between I42and β-Pix ( ). These observations indicate the involvement of Homer inthe function of I42/β-Pix and identify another signaling pathway thatcan be manipulated by agents that modulate Homer binding function.

ii) Ultrastructural localization of I42/β-Pix/Pak at synapses: We haveperformed preliminary immunoEM with I42 Ab and observes that it isassociated with the PSD region. The methods and approach are identicalto our studies of Shank (Naisbitt et al., 1999). This observationindicates that I42 is enriched at the excitatory synapse together withHomer, Shank and glutamate receptors.

Ryanodine Receptor (RYR) and Homer

The RYR encodes a potential Homer binding site near the N-terminus (Bhatet al., 1999) and using GST pulldown assays we observe that GSTHomerbinds to the relevant fragment of RYR1. Importantly, we havedemonstrated that the RYR co-immunoprecipitates with Homer fromdetergent extracts of skeletal muscle. The interaction between RYR andHomer is understood to be consistent with the function of Homer proteinsto regulate the coupling of membrane receptors with intracellularcalcium pools. Glutamate mediates an inhibitory postsynaptic potentialin dopamine neurons of the midbrain and this is mediated by mGluR1release of intracellular Ca2+ from RYR sensitive CICR pools (Bhat etal., 1999). RYR have recently been implicated as an important source ofNMDAR-induced calcium rise in the post synaptic spine (Emptage et al.,1999). Since Shank is part of the NMDA receptor signaling complex(Naisbitt et al., 1999) and binds Homer, it is compelling to evaluatethe possible interaction between RYR and Homer.

NMDA Receptor Type 2D (NR2D) and Homer

Independent Y2H screens of adult cortex and cerebellum identifiedseveral clones of the NMDA receptor type 2D (NR2D). NR2D has not been asextensively studied as NR2B but is expressed in developing cerebellumand interneurons in the forebrain (Dunah et al., 1998; Goebel andPoosch, 1999). NMDAR that include the NR2DR have slower channelproperties (Cull-Candy et al., 1998; Okabe et al., 1998; Vicini et al.,1998). The C-terminus of NR2D is highly proline rich consistent with ourobservation that Homer binds a specific proline rich sequence. Thus, inthe case of NR2D, Homer proteins form a direct coupling to CICR pools.This direct coupling would contrast with NMDAR that include NR2B whichappear to couple to Homer indirectly via PSD95-GKAP-Shank (Naisbitt etal., 1999). In both cases, modification of Homer crosslinking activitywill alter the intracellular release of calcium due to glutamatereceptor activation. Because of the differences in the bindingproperties of the EVH1 domain of Homer to its different targets, it isanticipated that agents that specifically disrupt the linkage of NR2B orNR2D can be developed.

Mammalian InaD like Molecule Interaction with Homer

We have identified two distinct novel members of a family of proteinswith similarity to the recently reported human InaD (Philipp andFlockerzi, 1997) and Drosophila Discs Lost DLT (Bhat et al., 1999).These proteins encode 5 and 4 PDZ domains, respectively, and a prolinerich region that is shared in all clones that is presumed to mediateinteraction with Homer. DLT has been demonstrated to be essential forestablishment of epithelial cell polarity and binds to the C-terminus ofNeurexin IV DLT (Bhat et al., 1999). We currently refer to our clones asrat InaD. In current studies, we observe that full length myc-taggedrInaD co-immunoprecipitates with Homer 2 from co-expressing HEK293cells.

I30 Interaction with Homer

I30 is a novel member of the family of abl binding proteins. Relatedproteins function as adaptor proteins that regulate cell growthZiemnicka-Kotula, 1998 #392; Biesova, 1997 #393 and are hypothesized .I30 encodes a SH3 domain and a Homer binding site. Accordingly, Homer isanticipated to link this protein to other Homer-interacting proteinsincluding metabotropic glutamate receptors and IP3R. (See SEQ ID NOS:15, 16, 19 and 20).

Cdc42-associated Tyrosine Kinase-2 (ACK-2) Interaction with Homer

ACK-2 is a non-receptor tyrosine kinase that is regulated by theRho-related GTP-binding protein Cdc42 Yang, 1999 #391. ACK-2 isactivated by signals that result from cell adhesion, by for exampleactivation of the integrin receptor. One cellular consequence of ACK-2activation is down stream activation of c-Jun kinase. Our observationthat ACK-2 interacts with Homer indicates that this signaling pathwaycan be linked to other membrane receptors by Homer, and identifiesanother signaling cascade that can be manipulated by agents that alterHomer crosslinking function.

EXAMPLE 1 Identification and Sequencing of Homer Family Members

Low stringency screens of phage cDNA libraries and EST Database searcheswere performed to identify Homer family members. cDNA libraries werescreened using the rat Homer 1a coding region as a probe. Screens ofmouse and rat brain cDNA libraries identified two isoforms of Homer-1(Homer-1b and Homer-1c).

Searches of EST Databases identified a mouse EST sequence (ID#442801)which is about 73% homologous to a portion of 5′ coding region ofHomer-1cDNA sequence. Based on the EST used RT-PCR (Forward: 5′-GAC AGCAGA GCC AAC ACC GTG-3′; (SEQ ID NO:49); Reverse: 5′-GTC TGC AGC TCC ATCTCC CAC-3′; (SEQ ID NO:50)) to amplify the corresponding region fromvarious mouse tissues. The PCR products (˜330 bp) consisted of twodifferent sequences, one of which contains an additional insertion of 33bp. A mixture of these two cDNA fragments were used as probes to screenan adult mouse brain cDNA library. Out of 10⁶ clones screened, fiveclones hybridized well to the probe. Sequence analysis of these clonesindicated that they are five partial cDNA clones representing twoisoforms of a Homer-2 gene. These clones are identical to the isoformsamplified by RT-PCR. The 5′ region of Homer-2 was cloned using 5′-RACEtechnique. Total RNA from E14.5 mouse brain was reverse-transcribedusing the reverse primer described above. Another gene-specific primer(5′-CAC GGT GTT GGC TCT GCT GTC-3′; (SEQ ID NO 51)) was used in theamplification of the 5′ region of Homer-2. The sequence authenticity ofthe 5′ RACE clones was further confirmed by sequencing a partial mouseEST clone #441857.

A search of the EST Database allowed the identification of several humanEST's corresponding to mouse and rat Homer-1b, Homer-2a and 2b cDNAsequences. RT-PCR was used to clone the human Homer-1b and Homer 2a and2b coding regions. A 5′ degenerate primer (5′-ATG GG(A/G/C) GA(A/G)CA(A/G) CC(T/C/G) AT(T/C) TTC-3′; (SEQ ID NO:52)) was designed based onan amino-terminal seven residue amino acid sequence (MGEQPIF; (SEQ IDNO:53)) that is conserved among human EST clone #HCE003, mouse, rat, andDrosophila Homer homologue sequences. The 3′ primers (5′-GAG GGT AGC CAGTTC AGC CTC-3′;_(SEQ ID NO:54)) for human Homer-1 and human Homer-2(5′-GTT GAT CTC ACT GCA TTG TTC-3′; (SEQ ID NO:55)) were made from thesequences of human EST clones #562862 and #HIBAB15 respectively. HumanHomer-1b and Homer-2a and 2b were amplified from new born human frontalcortex. The sequences of human Homer 1b, Homer 2a and Homer 2b werederived from sequencing several PCR clones and EST clones and are shownin SEQ ID NO's:3, 7 and 9.

Human and mouse Homer-3 were identified by searching EST Database, usingHomer-1 and Homer-2 sequences. Two full-length human Homer-3 clones wereidentified (Clone ID #284002 and #38753) and sequenced. Numerous mouseHomer-3 clones were found and one of them (Clone ID #1162828 ) containsan almost full-length coding region. Also identified were severalDrosophila EST sequences exhibiting significant homology at the aminoacid level to the N-terminal region of Homer family members. Thesequence presented in SEQ ID NO:11 is derived from Clone #LD3829.

Expression Constructs Mammalian expression constructs were made bycloning cDNA into SalI and NotI sites of pRK5 (Genentech), so that thecDNA was fused in-frame to an N terminal c-Myc tag. GST-fusionconstructs were made by cloning Homer cDNA into the SalI and NotI sitesof pGEX4T-2 (Pharmacia). The full-length coding regions of mouseHomer-1b, rat Homer-1c, mouse Homer-2b and human Homer-3 were engineeredwith SalI and NotI sites at the 5′ and 3′ ends by PCR using highfidelity DNA polymerase Pfu (Stratagene). Various truncations ofHomer-1b/c and Homer-2b coding regions were made by PCR with specificPrimers containing SalI and NotI sites. All the PCR-based constructswere sequenced to confirm the sequences and in-frame fusion.

The sequence of Homer 1a was used to screen cDNA libraries prepared fromrat and mouse brain for related gene products. Homer 1a sequence wasalso used to search GenBank data bases. Several related rodent and humansequences were identified.

cDNAs that are most closely related to Homer 1a appear to representalternative splice forms. This inference is based on nucleotide sequenceidentity of their 5′UTRs and the first 175 amino acids of the openreading frames (ORF). The presumptive novel splice variants, termedHomer 1b and 1c, are completely divergent from Homer 1a after residue175 of the ORF and they possess entirely distinct 3′UTRs. comparison atthe point of sequence divergence indicates that Homer 1a encodes aunique eleven amino acid carboxy terminus of the ORF and about 5 kb 3′UTR region. The unique eleven amino acid carboxy-terminal sequence ofHomer 1a does not possess a recognizable motif. In Homer 1b and 1c, anadditional 168 and 180 amino acids are present that are predicted topossess coiled-coil (CC) secondary structure (Lupas, Trends Biochem. Sci21:375 (1969)). While the 3′UTR sequence of Homer 1a includes multipleAUUUA repeats which are implicated in destabilizing mRNAs ofintermediate early genes (IEG) (Shaw and Kamen, Cell 46:659 (1986)), the3′UTR sequence of Homer 1b and 1c does not include this motif. The onlydifference between Homer 1b and 1c is the inclusion in Homer 1c of atwelve amino acid sequence insertion at residue 177, between theconserved amino-terminus and the CC domain. Thus, Homer 1b and 1c appearto be formed by a splicing event that substitutes a relatively long andunique carboxy-terminus of the ORF and shorter 3′UTR sequence that lacksthe characteristic IEG motif. Multiple independent isolates of rat andmouse Homer 1b and 1c were identified and sequenced to confirm theirnatural expression in brain.

Further searches identified cDNA sequences that appear to represent twoadditional Homer genes, termed Homer 2 and Homer 3. The sequences of twosplice forms of Homer 2 and one Homer 3 sequence is presented (SeeFigures section). The predicted size of the protein products and generaldomain structure are similar to Homer 1b and 1c. Like Homer 1b and 1c,each of the Homer 2 and Homer 3 proteins contain about 120 amino acidsat the amino-terminal that is highly similar to the amino-terminaldomain of Homer 1a . The degree of amino acid identity in these regionsis about 88% between Homer 1 and Homer 2 and about 86% between Homer 1and Homer 3. Many of the amino acid differences are conservative.

In contrast to the high degree of conservation in amino-terminal region,the carboxy-terminal regions of Homer 2 and 3 are only about 22%identical to Homer 1b, but like Homer 1b and 1c are predicted to possessa CC secondary structure. The CC domains of all Homer family membersexhibit significant homology (about 40-45% amino acid similarity) to theCC regions of myosin heavy chain (Strehler et al., J Mol Biol 190:291(1986)), kinesin heavy chain (Yang et al., Cell 56:879 (1989)) anddynactin (Gill et al, J. Cell Biol 115:1639 (1991)). The distinct spliceforms of Homer 2, termed Homer 2a and Homer 2b, are differentiated by aneleven amino acid insertion at residue 131 in Homer 2b. Human Homer 1, 2and 3 are mapped to chromosomes 5, 15 and 19, respectively by the HumanGenome Project.

Drosophila Homer possess the basic domain structure of mammalian Homers.The amino-terminus is highly homologous to that of mammalian Homer andthe carboxy terminus is predicted to form a CC secondary structure.

EXAMPLE 2 Generation and Characterization of Homer Antisera

Rabbit polyclonal antibodies were generated against synthetic peptidesderived from the unique carboxy termini of Homer 1b/c, Homer 2a/b andHomer 3. Synthetic carboxy-terminal peptides of Homer 1, 2 or 3 wereconjugated to thyroglobulin with glutaraldehyde and used to immunizerabbits according to a previously published protocol (Martin et al.,Neuron, 9:259 1992). Peptide sequences used are contained in Homer-1band 1c: IFELTELRDNLAKLLECS (SEQ ID NO:56); Homer-2a and 2b:GKIDDLHDFRRGLSKLGTDN (SEQ ID NO:57); and Homer-3: RLFELSELREGLARLAEAA(SEQ ID NO:58). Detergent (2% SDS) extracts from rat cortex,hippocampus, and cerebellum were separated on 8% SDS-PAGE gels andtransferred to nitrocellulose membranes. Blots was probed withpolyclonal anti-Homer sera. Specificity was tested by incubating theantiserum with 10 μg/ml of relevant peptide at room temperature for 10 mprior to use. Rabbit polyclonal antiserum was also generated against thefull length GST-Homer 1a fusion protein, as described previously(Brakeman, et al., Cell 87:227 1997). This antiserum recognizes allHomer 1 isoforms.

Unpurified antibodies were tested for their sensitivity and specificityin detecting heterologously expressed, full length Homer proteins withamino-terminal c-myc tags. Each Homer protein was selectively detectedon Western blot by the appropriate Homer antibody in soluble extracts oftransfected HEK293 cells. The myc-tagged Homer proteins migrated with anapparent molecular mass of 50 kDa. There was no cross reactivity betweenantibodies for one Homer form and other family members.

EXAMPLE 3 In Vitro Interaction of Homer Proteins with Cell-Surface mGluReceptors

To examine the interaction of Homer proteins with mGluR1 and mGluR5,HEK293 cells were transiently transfected (using calcium phosphate) withfull length mGluR1α and mGluR5 constructs in pRK5 (Brakeman et al.,1997). Cell lysates were made 24-48 h post-transfection. GST fusionproteins bound to glutathione agarose were prepared of Homer 1a , Homer1c, Homer 2b, Homer 3 and two amino terminal fragments of Homer 2according to the following procedure. GST fusion constructs wereprepared by polymerase chain reaction with specific primers thatincluded SalI and NotI sequences and subcloned into pGEX4T-2 vector(Pharmacia Biotech, Uppsala, Sweden). Constructs were confirmed bysequencing. GST-fusion proteins were expressed in BL21 bacterialstrains. Bacteria were harvested and lysed in PBS, 1% Triton X100, 2 mMphenylmethylsulfonyl fluoride (PMSF) and pelleted at 13,000 rpm (SorvallSS-34) at 4° C. for 5 m Proteins were purified by incubating 1 ml bedvolume glutathione-sepharose (GST) beads (Sigma USA) with bacterialsupernatant at 4° C. for 10 m, washing twice with PBS and PBS plus 1%Triton X-100. Protein was eluted with 10 mM glutathione and dialyzedagainst PBS at 4° C. Protein concentrations were measured by BCA(Pierce, Ill.). Cell lysates of the transfected cells were incubatedwith equivalent amounts of various Homer-GST fusion proteins at 4° C.for 2 h, washed with PBS and 1% Triton X-100. Proteins were eluted in 2%SDS sample buffer and separated on 8% or 2.5% SDS-PAGE gels and probedwith appropriate antibody.

It has been previously demonstrated that the amino-terminal 131 aminoacids of Homer 1a is sufficient to bind group I metabotropic glutamatereceptors (Brakeman et al., Nature 386: 284 (1997)). In view of the highdegree of sequence conservation in this region of Homer family members,the possibility that they would also bind group I receptors wasexamined. GST fusion proteins were prepared of Homer 1a, Homer 1 c,Homer 2b Homer 3 and two amino-terminal fragments of Homer 2. The fusionproteins were bound to glutathione agarose and assayed for binding tofull length mGluR5 or full length mGluR1a expressed in HEK293 cells.These studies show that mGluR5 bound GST Homer 1a . mGluR5 also bound toall full length Homer constructs and to a Homer 2 amino-terminalfragment of about 141 residues but not to GST alone. The relativebinding in the three assays were comparable for each of the three Homertypes. A Homer 2 deletion mutant that includes only the amino-terminal92 residues did not bind mGluR5. Similar binding of Homer proteins tomGluR1 was also observed.

EXAMPLE 4 In Vivo Interaction of Homer Proteins with Cell-Surface mGluReceptors

To examine if Homer proteins are naturally associated with group Imetabotropic receptors in the brain, immunoprecipitation studies wereperformed. Rat or mouse brain tissues were sonicated (3×10 s) in PBS(˜200 mg/ml wet weight) containing 1% Triton-X100 with proteaseinhibitors and centrifuged for 10 m at 15,000 g. Three μl of antiserumdirected against Homer 1b, Homer 1c, Homer 2a, Homer 2b or Homer 3 wasadded to 60 μl of tissue extract and incubated for 1½ h at 4° C. andthen washed three times with PBS/Triton. Preimmune and peptide-blockedantisera were used as negative controls. Binding in tissue samples wasanalyzed by gel electrophoresis and western blot analysis. Proteins wereeluted in 2% SDS loading buffer. mGluR1α monoclonal antibody wasobtained from PharMingen (San Diego Calif.). Rabbit polyclonal mGluR5antibody was a gift from Dr. Richard Huganir, Johns Hopkins School ofMedicine.

Homer family members are naturally associated with group I metabotropicreceptors in brain. This analysis was performed using cerebellum sinceall three Homer family members are expressed in this tissue. Detergentextracts of whole adult rat cerebellum were incubated with antibodies toHomer 1b/c. Homer 2a/b or Homer 3 and immunopreciptates were blottedwith a mouse monoclonal antibody to mGluR1α. mGluR1αco-immunoprecipitates with each of the antisera directed against Homerproteins. The predominate band after electrophoreses corresponded to themonomer form of mGluR1α (about 150 kDA) and other bands corresponding tomultimers of mGluR1α are also observed.

EXAMPLE 5 In Vitro Interaction of Homer Proteins with IntracellularInositol Trisphosphate Receptors

To demonstrate that Homer proteins interact in vivo with inositoltrisphosphate receptors immunoprecipitation studies were performed usingbrain tissue. Rats or mice were sacrificed by decapitation and thecerebella were dissected immediately. Cerebella were sonicated in TEbuffer (50 mM Tris, 1 mM EDTA, pH 7.4) containing 1% CHAPS and proteaseinhibitor cocktail (˜100 mg wet weight/ml). The homogenate wascentrifuged at 90,000 rpm, 20 m, 4° C. in a TLA 100.3 rotor. 100 μl ofthe cerebellar extract was used for each immunoprecipitation assay withthe following antibodies: 3 μl of crude Homer 1, Homer 2 or Homer 3antibodies (Xiao et al., in press); 20 μg of affinity purified inositoltrisphosphate antibody (gift from Alan Sharp). Antibodies and extractwere incubated for 30 m at 4° C., then 60 μl of 1:1 protein A or proteinG (for goat antibody) sepharose slurry was added. Theantibody/extract/beads were incubated for an additional 90 m at 4° C.After washing 3×10 m in TE-CHAPS buffer, the proteins were eluted fromthe beads with 30 μl of 4% SDS loading buffer and analyzed by SDS-PAGEand immunoblot.

Results from these studies showed that the inositol trisphosphatereceptor specifically co-precipitates with antisera directed againstHomer 1, Homer 2 and Homer 3.

EXAMPLE 6 Calcium Mobilization is Decreased by Transient Expression ofHomer Protein without a Coiled-Coil Domain

To demonstrate that Homer cross-links metabotropic glutamate receptorsand inositol trisphosphate receptors to provide or enhance a functionalsignaling complex, calcium mobilization was examined in cells transientexpressing truncated forms of Homer protein. The truncated Homer proteinused lacks the coiled-coil domain and is unable to form a bridge linkingthe mGluR at the cell surface with intracellular inositol trisphosphatereceptors. The truncated form of Homer protein resembled Homer 1a withthe exception of 11 residues at the carboxy-terminal. This form of Homerresults in enhanced expression of Homer protein as compared withtransfection of Homer 1a in heterologous cells. The Homer protein wasintroduced into Purkinje cells in primary cerebellar cultures andglutamate induced effects on calcium mobilization was measured.

Embryonic mouse cerebellar cultures were prepared and maintainedaccording to the method of Schilling et al. (Schilling et al., Neuron7:891 1991). At 4-5 DIV, cultures were transfected with plasmids codingfor E-GFP (Clontech) and either full-length Homer 1b or an IEG form ofHomer 1. The IEG form of Homer 1 was a 186 amino acid amino-terminalfragment of Homer 1b. Plasmids were purified by cesium banding. Threecombinations of the plasmids were transfected. Group I (control), 20 μgof E-GFP and 40 μg of pRK5 vector; group II, 20 μg of E-GFP and 40 μg ofpRK5 Homer 1 IEG; group III: 20 μg of E-GFP and 40 μg of pRK5 Homer 1b.Plasmid DNA was mixed with gold particles (0.6 micron), and coated ontoplastic tubing. DNA was then ballistically transfected into cellsaccording to the manufacturer's protocol (Helios Gene Gun System,BIO-RAD). After transfection, cultures were returned to the incubatorand maintained for an additional 2 days for a total of 7-8 DIV at thetime of use for imaging experiments.

Patch electrodes were attached to the somata of GFP-expressing Purkinjecells and a holding potential of −60 mV was applied. Micropressureelectrodes (1 μm tip diameter) were filled with quisqualate (100 μm inexternal saline) and were positioned ˜20 μm away from large-caliberdendrites. Test pulses were delivered using positive pressure (6 psi, 1sec). Cells were bathed in a solution that contained (in mM) NaCl (140),KCl (5), EGTA (0.2), MgCl₂ (0.8), HEPES (10), glucose (10), tetrodotoxin(0.005), and picrotoxin (0.1), adjusted to pH 7.35 with NaOH, whichflowed at a rate of 0.5 ml/m. The recording electrode contained CsCl(135), HEPES (10), fura-2 K₅ salt (0.2), and Na₂-ATP (4), adjusted to pH7.35 with C_(S)OH. Patch electrodes yielded a resistance of 3-5 MΩ whenmeasured with the internal and external salines described above.

Fura-2 ratio imaging of intracellular free Ca²+, was accomplished bymeasuring the background corrected fluorescence ratio at 340 and 380 nmexcitation using a cooled CCD camera system, as previously described(Linden et al., J Neurosci 15:5098 1995). Exposure times were 200 msecper single wavelength image. Experiments were conducted at roomtemperature. Enhanced GFP is weakly excited by illumination in the380-400 nm spectrum. Based upon the bandpass characteristics of our340HT15 and 380HT10 excitation filters and the absorption spectrum ofenhanced GFP (Clontech), we estimate that <1% of the signal at 340 nmexcitation and <5% of the signal at 380 nm excitation is contributed byGFP, even in those cells where the fura/GFP loading ratio is smallest.This could lead to a small (<5%) systemic underestimation of freecalcium concentration that should distribute randomly acrossexperimental groups.

Calcium mobilization in the absence of influx was measure by ratioimaging fura-2 in Purkinje cells bathed in Ca⁺²-free external saline andstimulated with a micropressure pulse of quisqualate, a metabotropicglutamate receptor agonist (Linden, Neuron 17:483 1996). The resultantCa⁺² transient is triggered by an mGluR and inositol trisphosphatepathway since it is completely blocked by either an mGluR antagonist((+)-MCPG, 500 μM in the bath) or a novel specific inositoltrisphosphate receptor-associated ion channel blocker, xestospongin C (1μM in the internal saline). Purkinje cells transfected with a truncatedform of Homer showed mGluR-evoked Ca⁺² responses with a decreasedamplitude (170±9 nM, mean±SEM, n=30 cells) and an increased latency(10.5±1.8 sec) as compared with cells transfected with Homer 1b (244±17nM, 4.2±0.9 sec, n=23) or an empty vector control (239±19 nM, 4.5±1.1sec, n=15). The decay phase of the Ca⁺² response appeared somewhatslower in neurons transfected with the truncated form. While the totalCa⁺² flux appeared similar in cells transfected with truncated andcomplete Homer proteins and in empty vector controls, the measurementcould not be made because the tail of the Ca⁺² response was abbreviateddue to the constraints of the image buffer capacity.

EXAMPLE 7 Determination of the Crystal Structure of Homer Protein

The crystal structure of Homer protein and a Homer protein binding sitewere determined. Results of these experiments are presented in Table

(a) Protein Expression and Purification

Residues 1-120 of rat Homer 1a were expressed in Escherichia coli BL21cells as a C-terminal fusion to glutathione-S-transferase (GST-laEVH) aspreviously described (Tu et al., Neuron 21:717 1998).Selenomethionine-substituted (SeMet) GST-1aEVH was prepared byexpression in the methionine auxotrophic strain B834 (DE3) (Novagen). 5mL of an overnight culture grown at 37° C. in LB media supplemented with100 μg/mL ampicillin (Sigma) was added to 4L M9 minimal media (GibcoBRL) supplemented with 100 μg/mL ampicillin, 0.05 mg/mL alanine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, tyrosine, L-selenomethione, 1μ/mLthiamine (Sigma), 2 mM MgSO₄, 1% glucose, 100 μM CaCl₂. Cells were grownto an A₆₀₀ of 0.5 at which time IPTG (Calbiochem) was added to a finalconcentration of 0.2 mM. Cells were grown for an additional 3 hours,harvested by centrifugation, and resuspended in 1×PBS/1% Triton.Pepstatin A and leupeptin (Boehringer-Mannheim) were added to a finalconcentration of 1 μg/mL, and PMSF (Life Technologies) was added to 0.5mM. Cells were lysed by sonication and centrifuged at 13,000 rpm in anSS-34 rotor to pellet cell debris. The cleared lysate was added to a 5mL glutathione-agarose (Sigma) column. The column was washed insuccession with twenty column volumes of 1×PBS/1% Triton, twenty columnvolumes of 1×PBS, and ten column volumes of cleavage buffer (50 mM Tris7.4, 150 mM NaCl, 2.5 mM CaCl₂, 50 mM β-mercaptoethanol). All bufferswere degassed. A 50% slurry of glutathione-agarose beads loaded withfusion protein was incubated with 20 U of biotinylated Thrombin(Novagen) for 16 h at room temperature. The released cleavage product(1a-EVH) was collected, and the biotinylated Thrombin was removed withstreptavidin-agarose beads (Novagen). 1a-EVH was further purified bycation-exchange chromatography using a Resource S column(Amersham-Pharmacia).

(b) Crystallization and Data Collection

Crystals of native and SeMet protein were grown in hanging drops by themethod of vapor diffusion (Wlodawer et al., Proc Natl Acad Sci USA72:777 1975). 1 μl of a 9 mg/mL native or SeMet protein solution wasmixed with a 1:1 dilution of reservoir buffer (30% PEG 3350, 87 mMMgSO₄, 50 mM HEPES, pH 7.3) with distilled water and equilibrated over ImL of reservoir buffer. All crystallization trials for the SeMet proteinwere set up under anaerobic conditions to minimize potential problemsdue to oxidation. Two different crystal forms were observed for both thenative and the SeMet protein. Crystals in the orthorhombic space groupP2₁2₁2₁, (unit cell dimensions a=33.79 Å, b=51.40 Å, c=66.30 Å)typically grew to a size of 0.5 mm×0.03 mm×0.03 mm. Crystals in thetrigonal space group P3₂21 (unit cell dimensions a=b=49.94 Å, c=80.91 Å)grew to a size of 0.4 mm×0.1 mm×0.1 mm. All data used for phasing andrefinement were collected from a single trigonal SeMet crystal soaked inmother liquor plus 10% (v/v) ethylene glycol for approximately threeminutes prior to flash freezing in a gaseous nitrogen stream at −180° C.X-ray diffraction data suitable for multiwavelength anomalous dispersion(MAD) phasing were collected at four wavelengths at or near the Seabsorption edge. These data were collected at beamline X4A of theNational Synchrotron Light Source at Brookhaven National Laboratoryusing an R-AXIS IV image plate detector. Nonoverlapping oscillations(2°) at φ and φ+180° were measured over a 90° rotation of the crystal,interleaving the four wavelengths. All data were processed and scaledusing the DENZO/SCALEPACK programs (Otwinowski and Minor, Meth Enzymol276:307 1997). Data collection statistics are shown in Table 1.

(c) Structure Solution and Refinement

The expected two selenium sites were determined and refined using theprogram SOLVE (Terwilliger and Berendzen, Acta Crystallogr D53:5711997;Terwilliger and Eisenberg, Acta Crystallogr A39:813 1983) and initial Sescattering factors from (Hall et al., Cell 91:85 1997). Values for therefined Se scattering factors as determined by SOLVE are shown inTable 1. The electron density maps calculated with the experimental MADphases as determined by SOLVE were improved by solvent flattening andhistogram matching using DM (Collaborative Computational Project, 1994).An initial model of residues 1-105 was built into 1.8 Å experimentalelectron density maps using the program O (Jones et al., ActaCrystallogr A47:110 1991). After one round of simulated annealing withbulk solvent correction and positional and B-factor refinement using CNS(Brünger et al., Acta Crystallogr D54:905 1998), residues 106-111 werebuilt into 2F₀-F_(c) maps. The model was refined against themaximum-likelihood target (Pannu and Read, Acta Crystallogr A52:6591996) using data to 1.7 Å Bragg spacing collected at 0.9879 Å. Eightrounds of model building and water addition alternated with B-factor andpositional refinement yielded the current model, which includes residues1-111 and 88 water molecules. No electron density was observed forresidues 112-120. This model has a crystallographic R value of 25.3% anda free R value of 28.4%. The solvent content is ca. 40.6%, with onemolecule per asymmetric unit. Fractional solvent accessibility for eachresidue was calculated in X-PLOR (Brünger, X-PLOR, Version 3.1: A systemfor X-ray crystallography and NMR (New Haven, Conn.: Yale Univ. 1992).

(d) Determination of Homer Site by Site Directed Mutagenesis

Point mutants of N-terminally myc-tagged, full-length Homer 1b and 1cand Homer 1 EVH1 were made using the QuikChange™ Site-DirectedMutagenesis Kit (Stratagene). Expression constructs were transientlytransfected into HEK293 cells using calcium phosphate methods. About24-48 h post-transfection, cell lysates were prepared in 1×PBS/1% TritonX-100 (Sigma) and protease inhibitors. GST pull-down assays wereperformed by mixing 100 μl of cell lysate with GST-mGluR5 or GST-Shank3(residues 1143-1408) (Tu et al., in press) bound to glutathione-agarose,incubating at 4° C. for 2 h, and washing with 1×PBS and 1×PBS/1% TritonX-100. Bound products were eluted with 100 μl 2×SDS loading buffer anddetected by SDS-PAGE and immunoblot using anti-myc antibody 9EI0(Invitrogen) and ECL reagents (Amersham).

EXAMPLE 8 Homer Expression is Upregulated in Certain Brain Regions inResponse to Electrically Induced Seizures

Rat Homer 1a was cloned based on its rapid upregulation in hippocampalgranule cell neurons following electrically-induced seizure (MECS; seeBrakeman et al., Nature 3:284 1997) The expression of other members ofthe Homer family was examined in the brain following seizure.Radio-labeled riboprobes were prepared using unique sequences for Homer1a, Homer 1b, Homer 1c, Homer 2a, Homer 2b and Homer 3. Probes used didnot distinguish between the splice forms of Homer 1b and 1c or Homer 2and 2b.

(a) In Situ Hybridization

Anti-sense and sense cRNA probes were generated from each mouse Homerplasmid by in vitro transcription in the presence of ³⁵SUTP, aspreviously described (Lyford et al., Neuron 14:433 1995). Probe forHomer-1a (Xiao, 1998; GenBank # AF093257) was derived from nucleotides1342 to 2140, for Homer 1b/c (Xiao, 1998; GenBank # AF093258) fromnucleotides 785 to 1396, for Homer-2a/b (Xiao, 1998; GenBank # AF093260submission) from nucleotides 486 to 1561, and for Homer-3 (GenBank #AF093261) from nucleotides 371 to 2123. Probe (about 10⁶ cpm in 75 μlhybridization buffer) was applied to each slide. Coverslipped slideswere then incubated in humidified chambers overnight at 56° C. Followingcompletion of wash steps, slides were air dried and exposed to KodakBiomax MR film for 2-3 days.

The anatomic distribution in unstimulated animals reveals thatexpression of Homer 1a is similar to the expression of Homer 1b andHomer 1c. High levels of expression of Homer 1a are observed in thehippocampus, striatum and cortex. In the cortex, there is laminarexpression with the highest levels in the superficial and deep layers.Expression of Homer 2a and 2b is enriched in the thalamus, olfactorybulb and principle neurons of the hippocampus in contrast to the cortexwhere low levels of expression of Homer 2a and 2b are observed. Homer 3is expressed primarily in the cerebellum and hippocampus.

In situ hybridization studies demonstrate the dramatic induction ofHomer 1a in response to MECS. In the hippocampus, induction ofexpression is estimated to be greater than 20-fold compared tohippocampus from unstimulated animals. MECS induced an increase in Homer1b and 1c expression of about 1.5 fold as determined by blot analysis.Expression of Homer 2 and Homer 3 is not altered in response to MECS.

EXAMPLE 9 Formation of Multimeric Complexes of Homer Proteins

The CC secondary structure is implicated in protein-protein interactions(Lupas, 1996 supra). Therefore, the possibility that this domain mightconfer the ability to form homo- or hetero-multimers between Homerfamily members was examined. For examining the coiled-coil interactionof Homer family members, myc-tagged Homer-1c and Homer-2b weretransfected into HEK293 cells and cell extracts were made 2-3 dayspost-transfection. Cell lysates were treated as described above.

First, the ability of full length, bacterially-expressed GST fusionproteins of Homer to bind full length myc-tagged Homer proteinsexpressed in HEK293 cells was tested. myc Homer 1c bound Homer 1b, Homer2b, Homer 3, Homer 1b and Homer 2b carboxy-terminal CC domain, but notHomer 1 a or Homer 2-amino-terminus. This is consistent with the notionthat the CC domain is important in the interaction, since Homer 1a andHomer 2-amino-terminus doe not encode the CC domain. To test thespecificity of the CC domain interactions, GST fusions of dynein IC-1aand dynein IC-2c were generated. The CC domains of these proteins showmodest sequence to Homer family CC domains and bind to the CC domain ofdynactin (Gill, 1991 supra). None of the myc-tagged Homer family membersbound to either dynein IC-1a or dynein IC-2c.

To determine whether Homer family members naturally form multimers inbrain, immunoprecipitates of cerebellum were examined. Extractsimmunoprecipitated with Homer 1b/c antibody contained Homer 3, whileextracts immunoprecipitated with Homer 3 contained Homer 1b/c. While itis possible that these co-immunoprecipitated Homer family members areassociated by means other than their CC domains, the fact theamino-terminus of Homer is monovalent and cannot form extendedconcatomers supports a model of multimerization mediated by the CCdomains. Homer 2 was not detected as a multimer with either Homer 1 orHomer 3 in these immunoprecipitation experiments.

EXAMPLE 10 Homer Family Proteins are Enriched at Brain SynapticFractions and are Expressed in Certain Peripheral Tissues

The distribution and localization of Homer family proteins was examinedat the using immunochemical methods. Tissue extracts were assayed usingimmunoblot analysis and tissue localization was examined usingimmunohistochemistry at the light and ultrastructural levels.

(a) Immunoblot Analysis

Immunoblot staining of SDS (2%) extracts of various brain regions wereexamined to assess the distribution of Homer proteins in the brain.Homer 1b/c antibody detected a single band of about 47 kDa in cortex,hippocampus and cerebellum. These regions have similar levels ofexpression. The Homer 2a/b antibody detected a single major band in eachof cortex, hippocampus and cerebellum. Less intense, higher apparentmolecular mass bands were detected at about 60 and about 80 kDa. Homer 3immunoblots showed low level expression in cortex and hippocampus andintense staining of a single band in cerebellum (47 kDa).Immmunostaining was completely blocked by preincubating the antibodywith 10 μl g/ml of the relevant peptide antigen.

(b) Immunohistochemistry

For light microscopy, rats were deeply anesthetized with sevoflurane andperfused through the aorta with 250 ml of saline followed by 400 cc eachof 4% paraformaldehyde in 0.1% phosphate buffer (pH 6.5) and 4%paraformaldehyde in 0.1% phosphate buffer (pH 8.5). The rat was allowedto postfix for 1 hr. at room temperature and then prefused with 15%sucrose in 0.1% phosphate buffer (pH 7.4). The brain was removed andsectioned at 40 μm on a freezing sliding block microtome and collectedin PBS. Tissue was stained with an immunoperoxidase technique, asfollows. Brain sections were incubated in PBS containing 0.3% H₂O₂ and0.25% Triton X-100 for 30 m and then washed 3×5 m in PBS. Sections wereincubated in a buffer “PGT” containing 3% normal goat serum (ColoradoSerum Co.) and 0.25% Triton X-100 in PBS for 1 hr. and then transferredto the primary antiserum diluted 1:750 in the same PGT buffer. Sectionswere gently shaken for 48 h at 4° C., washed 4×5 m in PBS and thenincubated for 1 hr. at room temperature in a goat anti-rabbit IgGconjugated to horseradish peroxidase (Biosource International) diluted1:100 in PGT. Sections were washed 4×5 m in PBS and incubated for 6 m atroom temperature in 0.05% diaminobenzidine dihydrochloride (DAB:Sigma)and 0.01% H₂O₂ in 0.1 M phosphate buffer. Sections were washed in PBS,mounted onto gelatin chrome-alum subbed slides, dehydrated in a seriesof graded ethanol, cleared in xylene and coverslipped with DPX (BDHLimited).

Immunohistochemistry was performed to determine the cellularlocalization of Homer 1b/c and Homer 2a/b and Homer 3 in rat brain.Light microscopic examinations indicated that all three Homer proteinsare enriched in Purkinje neurons. Immunoreactivity is present in thecytoplasmic region of the soma and extends prominently into thedendritic arbor. The nucleus is not stained. Little or no staining isdetected in the contiguous granule cell layer. A similar lightmicroscopic pattern of cellular localization was detected for Homer 3.Homer 2 immunostaining in cerebellum also showed staining in Purkinjeneurons, but appeared technically less differentiated.

(c) Electron Microscopy

For EM, a postembedding immunogold method as described previously (Wang,et al., J Neurosci 18:1148 1998) was used and modified from the methodof (Matsubara, et al., J Neurosci 16:4457 1996). Briefly, maleSprague-Dawley rats were perfused with 4% paraformaldehyde plus 0.5%glutaraldehyde in 0.1 M phosphate buffer. Two hundred micrometerparasagittal sections of the rostral cerebellum (folia III-V) werecryoprotected in 30% glycerol and frozen in liquid propane in a Leica EMCPC. Frozen sections were immersed in 1.5% uranyl acetate in methanol at−90° C. in a Leica AFS freeze-substitution instrument, infiltrated withLowicryl HM 20 resin at −45° C., and polymerized with UV light. Thinsections were incubated in 0.1% sodium borohydride plus 50 mM glycine inTris-buffered saline/0.1% Triton X-100 (TBST), followed by 10% normalgoat serum (NGS) in TBST, primary antibody in 1% NGS/TBST, 10 nmimmunogold (Amersham) in 1% NGS/TBST plus 0.5% polyethylene glycol, andfinally staining in uranyl acetate and lead citrate. Primary antibodieswere used at dilutions of 1:500 for Homer 1b and 1:100-1:400 for Homer3.

Immunogold EM of Purkinje neurons of the cerebellum was performed todetermine whether Homer family proteins are associated with synapticstructures. Homer 1b/c showed striking localization to the region of thepostsynapic spine. Gold particles are densely concentrated in the regionof the postsynaptic density (PSD). A very similar distribution is notedfor Homer 3 immunoreactivity. It is noted that rather than beingconcentrated directed over the PSD or the contiguous plasma membrane,the majority of the gold particles appear to be present in the cytoplasmimmediately subjacent to these structures.

Peripheral Tissues

Homer proteins are expressed in peripheral tissues. In detergentextracts of heart and kidney, a single band at 47 kDa immunoreactive toHomer 1b and 1c is detected. In extracts of liver, a complex of threebands ranging from about 44 to 47 kDa is detected. In heart, liver,skeletal muscle and intestine, bands immunoreactive to Homer 2a and 2bare detected. Homer 3 immunoreactive bands are detected in extracts oflung and thymus.

Subcellular Distribution

To examine the subcellular distribution of Homer proteins, a biochemicalfractionation of rat forebrain was performed and fractions were analyzedby Western blotting with Homer antibodies. Fractions were blotted formGluR5, BIP and synaptophysin to monitor anticipated enrichment offractions. Homer 1b/c, 2a/b and 3 were present in the crude nuclearpellet (P1), the medium spin crude synaptosomal pellet (P2), and thehigh speed microsomal pellet (P3). BIP is a 78 kDa ER resident protein(Munro and Pelham, Cell 48:899 (1987)). and was enriched in both the P3and the S3 fractions. While Homer 1b/c and Homer 3 were not abundant inthe soluble (S3) fraction, Homer 2 was enriched in the S3 fraction. TheP2 fraction was subfractionated after hypotonic lysis. The 25,000×gpellet (LP1), which is enriched in PSDs (Huttner et al., J Cell Biol96:1374 (1983)), showed enriched presence of mGluR5. The high speedpellet (165,000×g; LP2) showed the anticipated enrichment in thesynaptic vesicle protein synaptophysin (P38). Each of the Homer proteinswas enriched in the LP1 fraction relative to LP2. The final solublefraction (LS2) was uniquely enriched in Homer 2.

EXAMPLE 11 Transgenic Mouse Model Demonstrates that Expression of Homer1a Selectively Blocks Binding of Homer 1b/c to mGluR5 In Vivo

N-terminal myc-tagged full-length Homer 1 a ORF was cloned into theexpression vector pT2 (Gordon, et al., Cell 50:445 1987; Aigner, et al.,Cell 83:269 1995). Transgenic mice were generated at the University ofAlabama Transgenic Facility. Expression of the transgene protein wasassayed by western blot with rabbit polyclonal antisera that recognizesall Homer 1 isoforms (pan-Homer 1 antibody) and myc antibody.

Homer 1a is unique within the family of Homer related proteins in thatit is dynamically regulated and it lacks the CC domain. Accordingly, itwas hypothesized that the IEG would bind to group 1 metabotropicreceptors and disrupt the formation of multivalent complexes of Homerand mGluR. To examine this hypothesis, a transgenic mouse was generatedthat expresses Homer 1a under the control of a modified Thy-1 promoter(Gordon et al., 1987, supra), which drives neuron-specific expression inpostnatal brain (Aigner et al., 1995, supra). Transgenic mice expressedHomer 1a at high levels in cortex, hippocampus, cerebellum andthalamus/brainstem relative to levels in wild type litter mate controls.The pattern of Homer 1a transgene expression is consistent with thepreviously reported activity of this promoter (Gordon et al, 1987,supra). As expected, antibodies for both Homer 1b/c and Homer 2a/bco-immunoprecipitated mGluR5 from detergent extracts of wild typeforebrain. By contrast, Homer 1b/c antibody did notco-immunoprecipitates mGluR5 from transgenic mice. The effect of Homer 1a transgene expression was selective in that it did not disrupt theco-immunoprecipitation of Homer 3 with Homer 1b/c. The latterobservation is consistent with the notion that the Homer 1b/c-Homer 3interaction is mediated by the CC domain and is predicted not to bealtered by Homer 1a expression. Homer 1a was not part of the complexco-immunoprecipitated with Homer 1b/c, consistent with the notion thatthe CC is necessary for association with the complex. The effect of theHomer 1 a transgene in blocking the in vivo coupling of mGluR5 and Homer1b/c was additionally selective in that Homer 2 antibodyco-immunoprecipitated mGluR5 similarly from extracts of wild type andtransgenic mice. Thus Homer 1a appears to selectively disrupt theinteraction of Homer 1b/c with mGluR5 but not Homer 2 with mGluR5. Homer3 is less highly expressed in forebrain than Homer 1b/c or Homer 2a/band co-immunoprecipitates of mGluR5 with Homer 3 antibody were lessclean. Accordingly, it could not be determined in these experimentswhether Homer 1a also competes with Homer 3. Identical results wereobtained in tow independent mouse lines that express Homer 1a transgene.The Homer 1a expressing transgenic mice have not been behaviorallycharacterized but appear normal in size and gross motor activity.

EXAMPLE 12 Yeast Two-Hybrid Screen

To examine the physiological functions of Homer, a novel family ofproteins was identified based on its ability to interact with Homerfamily proteins in a yeast two-hybrid screen of a brain cDNA library.Homer 1a was subcloned into pPC97 (Chevray and Nathans, Proc. Natl.Acad. Sci. U.S.A., 89:5789 (1992)) and used to screen a random primedcDNA library prepared from seizure-stimulated rat hippocampus and cortexcloned in pPC86 (Chevray and Nathans, 1992, id.) as described previously(Brakeman et al., Nature, 386:284 (1997)). The same library wasrescreened using the PDZ domain of Shank 3 (amino acid residues 559-673)cloned into pPC86. The Shank 3 PDZ domain was also tested forinteraction with mGluR constructs in pPC86. mGluR5 constructs included awild type C-terminal 241 amino acid fragment and a four amino acidcarboxy-terminal deletion of the same fragment.

Using Homer as “bait” in a yeast two-hybrid screen of a rat cortex andhippocampus cDNA library, multiple cDNA isolates of two novel genes wereobtained. Sequencing and full length cloning identified these asdistinct members of a gene family, termed Shank 1 and 3 (Naisbitt etal., Neuron (1999) 23:569-82). Shank family proteins are closely relatedto a previously described protein, termed Cortactin Binding protein(CortBP-1; Du et al., Mol. Cell. Biol., 18:5838 (1998)).

EXAMPLE 13 Interactions Between Homer Proteins and Shank Proteins InVitro and In Vivo

To characterize the interaction between Homer proteins and Shankproteins, the Shank cDNAs isolated from the yeast two-hybrid screen(Example 10)) were expressed in HEK293 for GST pulldown assays withGST-Homer 1a. The interaction between Homer and Shank proteins wasfurther characterized by co-immunoprecipitation assays.

(a) Expression Constructs

Expression constructs were transiently transfected into HEK293 cellsusing the calcium phosphate method. Cells were lysed 24-48 hpost-transfection with PBS plus 1% Triton X-100. GST pull down assayswere performed by mixing 100 μl cell lysates with beads charged with GSTfusion proteins (1-3 μg/50 μl bed vol.) at 4° C. for 2 h followed bywashing once with PBS, once with PBS plus 1% Triton X-100. Boundproteins were eluted with 100 μl 2×SDS loading buffer and detected bySDS-PAGE and immunoblotting using ECL reagents (Amersham). GST pull downassays of mGluR1a and mGluR5 from brain lysates were performed bysonicating rat cerebellum or cortex in 50 mM Tris, 1 mM EDTA, 1% CHAPS(Sigma), 0.5% deoxycholic acid (Sigma) and proteinase inhibitors withGST-proteins and these tissue extracts were then processed as above. Forimmunoprecipitation from COS7 cells, transfected cells were extracted inRIPA (see Naisbitt et al., 1999, supra). Soluble extracts wereprecipitated with 2 μg control non-immune IgG, Myc or Shank 1 (56/e)antibodies (Naisbitt et al., 1999, supra).

(b) GST Pulldown and Co-immunoprecipitation Assays

Expression constructs were transiently transfected into HEK293 cellsusing the calcium phosphate method. Cells were lysed 24-48 hpost-transfection with PBS plus 1% Triton X-100. GST pull down assayswere performed by mixing 100 μl cell lysates with beads charged with GSTfusion proteins (1-3 μg/50 μl bed vol.) at 4° C. for 2 h followed bywashing once with PBS, once with PBS plus 1% Triton X-100. Boundproteins were eluted with 100 μl 2×SDS loading buffer and detected bySDS-PAGE and immunoblotting using ECL reagents (Amersham). GST pull downassays of mGluR1a and mGluR5 from brain lysates were performed bysonicating rat cerebellum or cortex in 50 mM Tris, 1 mM EDTA, 1% CHAPS(Sigma), 0.5% deoxycholic acid (Sigma) and proteinase inhibitors withGST-proteins and these tissue extracts were then processed as above. Forimmunoprecipitation from COS7 cells, transfected cells were extracted inRIPA (see Naisbitt et al., in press). Soluble extracts were precipitatedwith 2 μg control non-immune IgG, Myc or Shank 1 (56/e) antibodies(Naisbitt et al., in press).

Extracts of forebrain crude synaptosomes for immunoprecipitation wereprepared using deoxycholic acid as described previously (Dunah et al.,Mol. Pharmacol. 53429 (1998)). Forebrain P2 fraction was extracted in 1%deoxycholic acid, dialyzed over night into 0.1% Triton X-100, 50 mMTris, pH 7.4. Concurrently, 5 g of each antibody was pre-incubatedovernight with 10 μl bed volume protein A-sepharose. Aftercentrifugation at 100,000 g for 1 h, 50 μg of extract was incubated withantibody-protein A in 100 μl 0.1% Triton X-100, 50 mM Tris, pH 7.4 for 2h at 4° C. Pellets were washed 4 times with 1 ml incubation buffer, andbound proteins were analyzed by immunoblotting.

Antibodies Shank antibodies were raised in rabbits immunized withGST-fusions of Shank 3 residues 1379-1740 and 1379-1675 (Covance,Denver, Pa.). Similar bands were seen on rat brain immunoblots with bothantisera. GKAP, PSD 95 and Shank 1 (56/e) antibodies are described in(Naisbitt et al., 1999, supra). Homer antibodies are described above.Anti-mGluR 1a monoclonal antibody is from Pharmingen and rabbitpolyclonal mGluR5 antiserum was obtained from Dr Richard Huganir (JohnsHopkins University).

Shank cDNAs derived from the yeast two-hybrid screen were expressed inHEK293 cells for GST pulldown assays with GST-Homer 1a. Each of theShank polypeptides specifically bound Homer 1a. Based on the findingthat the Homer EVH1 domain binds a specific proline-rich motif, threepotential Homer binding sites (or Homer “ligands”) that are conserved inShank 1, 2, 3 and CortBP-1 were identified. (Naisbitt et al., 1999,supra). To define the Homer binding site on Shank family proteins, threedeletion fragments of Shank 3 that included, respectively, amino acidresidues 559-908, amino acid residues 1143-1408, and amino acid residues1379-1740 were testing for their ability to bind to Homer 1b, Homer 1c,Homer 2 and Homer 3 in GST pulldown assays. Similar binding specificitywas detected with each of the Homer proteins. Only Shank3 fragment1143-1408 bound to Homer. This region contains the amino acid sequencethat most closely resembles the Homer ligand peptide consensus(LVPPPEEFAN; residues 1307-1316). A similar sequence is present inShank1 (PLPPPLEFSN 1563-1572; see Naisbitt et al., 1999, supra). CortBPpossesses two similar sites; (PLPPPLEFAN; residues 813-822) and(FLPPPESFDA residues 878-887). Fragments of Shank3 containing amino acidresidues located nearer the amino-terminal of the protein such as Shank3 fragment 559-908 (which includes the PDZ domain and the firstproline-rich motif) did not bind to Homer, but did bind to GKAP(Naisbitt et al., 1999, supra). Similarly, Shank3 fragment 1379-1740,which includes the carboxy-terminal proline-rich sequence and the SAMdomain, did not bind to Homer, though it is capable of binding itselfand cortactin (Naisbitt et al., 1999, supra). These studies identify theHomer binding site as being distinct from either the PDZ domain thatbinds GKAP, or the proline-rich binding site that binds cortactin andwhich is located nearer to the carboxy-terminal (Naisbitt et al., 1999,supra).

To confirm the site of Homer interaction, site directed point mutants ofthe putative Homer ligand in Shank3 were assessed for their ability tobind to GST-Homer 1c. Full length wild type Shank 3, Shank3(P1311L), andShank3(F1314C) were expressed in HEK293 cells and assayed for binding toGST-Homer 1c. Compared to wild type Shank 3, both point mutants showeddramatically reduced binding to Homer., These experiments providefurther confirmation that the Homer ligand in Shank3 is the principlesite of interaction.

It has been previously demonstrated that amino acids 1-110 of the HomerEVH1 domain are necessary and sufficient for binding to Homer ligands(Brakeman et al., 1997, supra; Tu et al., 1998, supra). To confirm thatthe EVH1 domain of Homer mediates interactions with Shank, a series ofpoint mutants of the Homer 1 EVH1 domain were generated. Mutations thatdisrupted binding to mGluR5 disrupted binding to Shank 3 in an identicalmanner, indicating Homer binds both proteins via a similarEVH1-dependent mechanism (Beneken et al., 2000, supra).

To confirm the interaction between Homer and Shank in a mammalian cellcontext, co-immunoprecipitation experiments were performed inheterologous cells. COS7 cell were transfected with Myc tagged-Homer 1b,Shank 1, or Shank 1 plus myc-Homer 1b. Detergent extracts of cells weresubjected to immunoprecipitation and blotted with myc, shank, or control(non-immune IgG) antibodies. Homer 1b was used in these experimentsbecause it expresses more efficiently in mammalian cells than Homer 1a.There is co-immunoprecipitation of Homer with Shank antibody and ofShank with myc antibody only from cells expressing both Shank andmyc-Homer 1b.

To demonstrate the in vivo relevance of the Homer-Shank interaction,co-immunoprecipitation experiments were performed using detergentextracts of rat brain. Detergent extracts of rat forebrain fractionswere immunoprecipitated with Shank and control (non-immune) antisera.Immunoprecipitates were blotted for Homer, Shank and GRIP antibodies.Antibodies raised against a fusion protein of Shank 1 immunoprecipitatedHomer 1b and 1c proteins as well as Shank from rat forebrain. GRIP wasnot co-immunoprecipitated with Shank and neither Shank or Homer wereprecipitated by non-immune IgGs. Furthermore, another Shank antibody,generated against Shank 3 fragment 1379-1675, co-immunoprecipitatedHomer 1b and 1c extracted from both cerebellum and cortex.

EXAMPLE 14 Homer and Shank Mediate Clustering of Cell-Surface Receptors

Shank proteins may link Homer proteins with components of a cell-surfaceclustering complex, such as the NMDA clustering complex.

COS7 cells were transfected using the Lipofectamine method (GIBCO-BRL)on poly-lysine coated coverslips for clustering experiments, asdescribed in Naisbitt et al. ([in press] 1999, supra) and Kim et al.(Neuron 17:103 1996). Primary antibodies were used as follows: GKAPC9589, 1 μg/ml (Naisbitt et al., 1999, supra); Shank 56/e 0.5 μg/ml(Naisbitt et al., 1999, supra), PSD-95, 1:1000 diluted guinea pig serum(Kim et al., Neuron 378:85 1995). Cy3 and (fluoroscein isothiocyanateconjugate (FITC)- conjugated secondary antibodies (JacksonImmunoresearch) were used at dilutions of 1:500 and 1:100 respectively.

Yeast two-hybrid screens were performed as described in Example 10.

A yeast two-hybrid screen of the same rat brain cDNA library wasperformed using the PDZ domain of Shank3 as bait. From this screen, twoidentical clones of the carboxy-terminus of GKAP-3/SAPAP3 were isolated.In a reciprocal screen, Naisbitt et al., 1999, supra) isolated multipleclones of Shank1, 2 and 3 using GKAP as bait. This result providesindependent confirmation of the specificity of the interaction betweenthe Shank and GKAP/SAPAP families of proteins.

The cDNA from the yeast two-hybrid screen encoding the carboxy-terminal347 amino acids of GKAP-3 was expressed with an amino-terminal myc tagin HEK293 cells and tested for binding to GST fusion constructs ofShank3 and other PDZ containing proteins. The GST fusion of Shank3fragments containing just the PDZ domain (residues 559-673) wassufficient to bind GKAP3, while a Shank3 construct lacking the PDZdomain (residues 665-908) failed to bind. Additionally, PDZ domains ofGRIP and SAP102 failed to pull down GKAP3, demonstrating the specificityof the Shank-GKAP interaction.

The above findings suggest that Homer, Shank and GKAP may assemble intoa ternary complex. To explore this further, GST pull-down assays wereperformed using rat brain extracts. The carboxy-terminal 76 amino acidsof GKAP 1a, containing the Shank PDZ-binding sequence -QTRL, was fusedto GST GST-GKAP(carboxy-terminal). GST-GKAP(carboxy-terminal)specifically pulled down both Shank and Homer 1b and 1c, but not GKAP1or several other proteins (Naisbitt et al., 1999, supra). Since GKAPbinds directly to Shank but not to Homer (Naisbitt et al., 1999, supra),the results suggest that the GKAP pulldown of Homer is mediated byShank. These findings corroborate the co-immunoprecipitation experimentsof Shank and Homer from brain extracts and confirm that Homer isassociated with Shank in a native complex.

Since Shank proteins may link Homer proteins with components of the NMDAclustering complex, co-clustering of these proteins in transfected COScells was assessed. In cells co-expressing Homer 1b and PSD-95, bothproteins showed a diffuse distribution in the cytoplasm. This is notsurprising, since Homer and PSD-95 do not interact directly. When cellswere transfected with Shank1 and GKAP in addition to Homer and PSD-95,Homer and PSD-95 redistributed into plaque-like clusters in which bothproteins were exactly co-localized. By contrast, co-clustering of Homerand PSD-95 was not observed following co-transfection of Homer andPSD-95 with either Shank1 or GKAP alone. Thus, Homer and PSD-95co-cluster only upon co-expression of Shank and GKAP. Therefore, Shankand GKAP may mediate the formation of a quaternary protein complexcontaining PSD-95 and Homer (see also Naisbitt et al., 1999, supra).Other types of macromolecular complexes may also form when Homer andShank proteins interact. Cells expressing Homer 1b and Shank 1 (withoutGKAP or PSD-95) exhibited a redistribution of Homer 1b into a reticularfilamentous pattern, as well as into clusters; in both kinds ofstructures Shank and Homer immunoreactivities were co-localized. Thesefindings provide further evidence for an interaction between Homer andShank, and suggest that Homer 1b and Shank can co-assemble into higherorder macrocomplexes. This result is consistent with the biochemicalproperties of Shank that include its ability to self-multimerize andbind cortactin (Naisbitt et al., 1999, supra). Since Shank, GKAP, andPSD-95 are components of NMDA receptor-associated complex (Naisbitt etal., 1999, supra), the identification of Homer as a Shank-bindingprotein invokes a molecular link between the NMDA receptor complex andHomer-associated synaptic proteins such as mGluR1a and 5 and theinositol trisphosphate receptor.

Group 1 Metabotropic Receptors

Based on the observations in heterologous cells that Shank clusters withHomer 1b and that Shank together with GKAP can mediate the co-clusteringof Homer and PSD-95 Shank may mediate clustering of group 1 metabotropicglutamate receptors (mGluRs). Co-expression of Shank1 and mGluR5 in COScells did not result in obvious clustering of either protein. Similarly,Homer and mGluR5 do not form co-clusters. Co-expression of the threeproteins Homer, Shank 1, and mGluR5, however, resulted in conspicuousco-clustering of mGluR5 with Shank 1. Clustering of mGluR5 in thesetriply transfected cells was dependent on the ability of Homer to bindthe receptor since a point mutant of mGluR5 that does not interact withHomer failed to co-cluster with Shank. Thus, both Homer and Shank arerequired to mediate the clustering of mGluR5.

EXAMPLE 15 The Shank 3 PDZ Domain Binds the Carboxy-Terminus of Group 1Metabotropic Receptors Directly at a Site Distinct from the HomerBinding Site

The Shank PDZ domain shows selective binding to the GKAPcarboxy-terminus (Naisbitt et al., 1999, supra). The carboxy-terminalsequence of GKAP (-QTRL) finds similarities with that of the group 1mGluRs (mGluR1a -SSSL; mGluR5 -SSTL) and therefore it was determinedwhether the PDZ domain of Shank can directly bind the carboxy-terminusof group 1 mGluRs. GST-pulldown assays were performed using extractsfrom heterologous cells expressing a recombinant mGluR5 carboxy-terminal241 amino acid peptide. The mGluR5 carboxy-terminal tail bound twopartially overlapping constructs of Shank 3 that included the PDZ domain(559-908; and 559-673), but not a construct from which the PDZ domainwas deleted (amino acids 665-908). Binding of mGluR to the Shank3 PDZdomain was qualitatively similar to mGluR5 binding to Homer 1c and Homer2. Negative controls included absence of binding of mGluR to SAP102PDZ1-3 and GRIP PDZ 4-6. Furthermore, a deletion mutant of the mGluR5polypeptide that lacked the carboxy-terminal four amino acids failed tobind to the PDZ domain of Shank3. Identical interactions between ShankPDZ and mGluR5 C-terminal tail were detected in a yeast two-hybridanalysis. These studies indicate that the PDZ domain of Shank 3 can bindthe carboxy-terminus of group 1 metabotropic receptors via aPDZ-mediated interaction with the carboxy-terminal sequence —S S/T L.

To confirm that Shank3 PDZ domain can bind full length native mGluRs,GST pull down assays were performed with detergent extracts of forebrainor cerebellum. The PDZ domain of Shank 3 bound specifically to mGluR1aand mGluR5 from cerebellum and forebrain, respectively. (Cerebellumpredominantly expresses mGluR1, while forebrain expresses predominantlymGluR5.) While it is possible that the Shank3 PDZ pulldown of mGluRsfrom brain extracts is indirect, via Shank PDZ pulling down aGKAP-Shank-Homer-mGluR complex, this extended complex is unlikely giventhe more modest ability of GST-GKAP to pull down Homer.

These studies suggest that Shank may interact with the cytoplasmic tailof mGluR1a/5 both directly, via its PDZ domain, and indirectly, viaHomer. The inability of Shank 1 to cluster mGluR5 in the absence ofHomer indicates that the direct PDZ-dependent Shank-mGluR interaction iscontingent upon a co-incident Homer interaction. Both modes ofinteraction with mGluR may be involved in mGluR clustering by Shank andcontribute to physiological regulation.

EXAMPLE 16

Shank and Homer Co-Localization at Specific Post Synaptic Densities

Immuno Electron Microscopy A postembedding immunogold method (Petraliaet al., Nature Neurosci 2:31 1999; Zhao et al., J Neurosci 18:5517 1998)was used. Male Sprague-Dawley rats was perfused with 4% paraformaldehydeplus 0.5% glutaraldehyde in 0.1 M phosphate buffer (PBS). Parasagittalsections (250 μm) of the hippocampus were cryoprotected in 30% glyceroland frozen in liquid propane in a Leica EM CPC. Frozen sections wereimmersed in 1.5% uranyl acetate in methanol at −90° C. in a Leica AFSfreeze-substitution instrument, infiltrated with Lowicryl HM 20 resin at−45° C., and polymerized with UV light. Thin sections were incubated in0.1% sodium borohydride plus 50 mM glycine in Tris-buffered saline/0.1%Triton X-100 (TBST), followed by incubations in 10% normal goat serum(NGS) in TBST, primary antibody in 1% NGS/TBST, 10 nm immunogold(Amersham) in 1% NGS/TBST plus 0.5% polyethylene glycol, and finallystaining with uranyl acetate and lead citrate. For double labeling, thefirst primary antibody (e.g., Shank; Shank3 1379-1675 antigen) andcorresponding immunogold-conjugated antibody (10 nm gold) were applied,sections were exposed to paraformaldehyde vapors at 80° C. for one hour,and the second primary (Homer 1b and 1c) and secondary (20 nm gold; TedPella/BBI International) antibodies were applied the following day.Controls (showing little or no gold labeling) included absence of theprimary antibody for single labeling and absence of the second primaryantibody for double labeling. Primary antibodies were used at dilutionsof 1:100-1:300 for Shank and 1:400 for Homer 1b and 1c.

An antibody generated against a carboxy-terminal region of Shank 3(amino acids 1379-1675) was used to examine the ultrastructuraldistribution of the Shank proteins in brain. This antibody recognizesmultiple bands on brain immunoblots, including major bands of ˜160-180kD and ˜210 kD in forebrain and cerebellum, similar to those seen withother Shank antibodies (see Naisbitt et al., 1999, supra). The differentsize bands presumably derive from the multiple Shank genes and splicevariants. All Shank immunoreactivity is blocked by incubation of theShank antibody with the Shank fusion protein antigen.

Immunogold electron microscopy revealed intense Shank immunoreactivityat the PSD of CA1 pyramidal neurons. Gold particles were distributedover the entire region of the PSD. In the same preparations, Homer 1b/1cwas found to co-distribute with Shank. In all profiles withimmunostaining for both Shank and Homer, gold particles were presentover the PSD but also extended into the region subjacent to the PSD.This distribution is similar to the distribution of NMDA receptorsassociated with the postsynaptic membrane (Petralia et al., 1999, supra)and distinct from the distribution of mGluR5 which are most prevalent inthe perisynaptic membrane region just outside the PSD (Lujan et al., EurJ Neurosci 8:1488 1996). This spatial localization is consistent withthe idea that Shank 3 and Homer interact with components of both theNMDA receptor and metabotropic receptor signaling complexes.

This family of proteins that interact with Homer are identical to theShank family of postsynaptic density (PSD) proteins that interact withGKAP and PSD-95 complex (Naisbitt et al., 1999, supra). Shank usesdistinct domains to bind to GKAP and to Homer, and thus can form abridge between proteins of this family. Shank/GKAP is also associatedwith NMDA receptors through the PSD-complex (Naisbitt et aL, 1999,supra) and thus the Homer-Shank interaction indicates a molecular linkbetween NMDA receptors and Homer-associated proteins such as mGlureceptors and inositol trisphosphate receptors. This linkage hasimportant implications for the coupling of NMDA receptors tointracellular calcium release pools and for excitatory synapse assemblyin general.

REFERENCES CITED

Allen, K. M., Gleeson, J. G., Bagrodia, S., Partington, M. W.,MacMillan, J. C., Cerione, R. A., Mulley, J. C., and Walsh, C. A.(1998). PAK3 mutation in nonsyndromic X-linked mental retardation. NatGenet 20, 25-30.

Bagrodia, S., Taylor, S. J., Jordon, K. A., Van Aelst, L., and Cerione,R. A. (1998). A novel regulator of p21-activated kinases. J Biol Chem273, 23633-6.

Biesova, Z., Piccoli, C., and Wong, W. T. (1997). Isolation andcharacterization of e3B1, an eps8 binding protein that regulates cellgrowth. Oncogene 14, 233-41.

Yang, W., Lin, Q., Guan, J. L., and Cerione, R. A. (1999). Activation ofthe Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion viaintegrin betal. J Biol Chem 274, 8524-30.

Ziemnicka-Kotula, D., Xu, J., Gu, H., Potempska, A., Kim, K. S.,Jenkins, E. C., Trenkner, E., and Kotula, L. (1998). Identification of acandidate human spectrin Src homology 3 domain- binding protein suggestsa general mechanism of association of tyrosine kinases with thespectrin-based membrane skeleton. J Biol Chem 273, 13681-92.

Abel, T., Nguyen, P. V., Barad, M., Deuel, T. A., Kandel, E. R., andBourtchouladze, R. (1997). Genetic demonstration of a role for PKA inthe late phase of LTP and in hippocampus-based long-term memory. Cell88, 615-26.

Aiba, A., Chen, C., Herrup, K., Rosenmund, C., Stevens, C. F., andTonegawa, S. (1994). Reduced hippocampal long-term potentiation andcontext-specific deficit in associative learning in mGluR1 mutant mice.Cell 79, 365-75.

Aigner, L., Arber, S., Kapfhammer, J. P., Laux, T., Schneider, C.,Botteri, F., Brenner, H. R., and Caroni, P. (1995). Overexpression ofthe neural growth-associated protein GAP-43 induces nerve sprouting inthe adult nervous system of transgenic mice. Cell 83, 269-78.

Allen, K. M., Gleeson, J. G., Bagrodia, S., Partington, M. W.,MacMillan, J. C., Cerione, R. A., Mulley, J. C., and Walsh, C. A.(1998). PAK3 mutation in nonsyndromic X-linked mental retardation. NatGenet 20, 25-30.

Aniksztejn, L., Bregestovski, P., and Ben-Ari, Y. (1991). Selectiveactivation of quisqualate metabotropic receptor potentiates NMDA but notAMPA responses. Eur J Pharmacol 205, 327-8.

Arai, I., Shimazoe, T., Shibata, S., Inoue, H., Yoshimatsu, A., andWatanabe, S. (1996). Enhancement of dopamine release from the striatumthrough metabotropic glutamate receptor activation in methamphetaminesensitized rats. Brain Res 729, 277-80.

Arai, I., Shimazoe, T., Shibata, S., Inoue, H., Yoshimatsu, A., andWatanabe, S. (1997). Methamphetamine-induced sensitization of dopaminerelease via a metabotropic glutamate receptor mediated pathway in ratstriatal slices. Jpn J Pharmacol 73, 243-6.

Bagrodia, S., Taylor, S. J., Jordon, K. A., Van Aelst, L., and Cerione,R. A. (1998). A novel regulator of p21-activated kinases. J Biol Chem273, 23633-6.

Banno, T., and Kohno, K. (1998). Conformational changes of the smoothendoplasmic reticulum are facilitated by L-glutamate and its receptorsin rat Purkinje cells. J Comp Neurol 402, 252-63.

Barnes, C. A., Jung, M. W., McNaughton, B. L., Korol, D. K., Andreasson,K., and Worley, P. F. (1994). LTP saturation and spatial learningdisruption: Effects of task variables and saturation levels. Journal ofNeuroscience 14, 5793-5806.

Bashir, Z. I., Bortolotto, Z. A., Davies, C. H., Berretta, N., Irving,A. J., Seal, A. J., Henley, J. M., Jane, D. E., Watkins, J. C, andCollingridge, G. L. (1993). Induction of LTP in the hippocampus needssynaptic activation of glutamate metabotropic receptors. Nature 363,347-50.

Baude, A., Nusser, Z., Roberts, J. D., Mulvihill, E., McIlhinney, R. A.,and Somogyi, P. (1993). The metabotropic glutamate receptor (mGluR1alpha) is concentrated at perisynaptic membrane of neuronalsubpopulations as detected by immunogold reaction. Neuron 11, 771-87.

Ben-Ari, Y., Aniksztejn, L., and Bregestovski, P. (1992). Protein kinaseC modulation of NMDA currents: an important link for LTP induction.Trends Neurosci 15, 333-9.

Beneken, J., Tu, J. C., Xiao, B., Yuan, J. P., Worley, P. F., and Leahy,D. J. (submitted). Crystal structure of the Homer EVH1 Domain: Aversitile binding module with unexpected homology to PH domains. Neuron.

Berke, J. D., Paletzki, R. F., Aronson, G. J., Hyman, S. E., and Gerfen,C. R. (1998). A complex program of striatal gene expression induced bydopaminergic stimulation. J Neurosci 18, 5301-10.

Berridge, M. J. (1998). Neuronal Calcium Signaling. Neuron 21, 13-26.

Bhat, M. A., Izaddoost, S., Lu, Y., Cho, K. O., Choi, K. W., and Bellen,H. J. (1999). Discs Lost, a novel multi-PDZ domain protein, establishesand maintains epithelial polarity. Cell 96, 833-45.

Blue, M. E., and Parnavelas, J. G. (1983). The formation and maturationof synapses in the visual cortex of the rat. I. Qualitative analysis. JNeurocytol 12, 599-616.

Bonci, A., and Williams, J. T. (1996). A common mechanism mediateslong-term changes in synaptic transmission after chronic cocaine andmorphine. Neuron 16, 631-9.

Bootman, M. D., Berridge, M. J., and Lipp, P. (1997). Cooking withcalcium: the recipes for composing global signals from elementaryevents. Cell 91, 367-73.

Bortolotto, Z. A., Bashir, Z. I., Davies, C. H., and Collingridge, G. L.(1994). A molecular switch activated by metabotropic glutamate receptorsregulates induction of long-term potentiation. Nature 368, 740-3.

Brakeman, P. R., Lanahan, A. A., O'Brien, R., Roche, K., Barnes, C. A.,Huganir, R. L., and Worley, P. F. (1997). Homer: a protein thatselectively binds metabotropic glutamate receptors. Nature 386, 284-8.

Carlezon, W. A., Jr., Thome, J., Olson, V. G., Lane-Ladd, S. B.,Brodkin, E. S., Hiroi, N., Duman, R. S., Neve, R. L., and Nestler, E. J.(1998). Regulation of cocaine reward by CREB. Science 282, 2272-5.

Chevray, P. M., and Nathans, D. (1992). Protein interaction cloning inyeast: identification of mammalian proteins that react with the leucinezipper of Jun. Proc Natl Acad Sci U S A 89, 5789-93.

Cole, A., Saffen, D., Baraban, J., and Worley, P. (1989). Rapid increaseof an immediate early gene mRNA in hippocampal neurons by synaptic NMDAreceptor activation. Nature 340, 474-476.

Conquet, F., Bashir, Z. I., Davies, C. H., Daniel, H., Ferraguti, F.,Bordi, F., Franz-Bacon, K., Reggiani, A., Matarese, V., Conde, F., andet, a. (1994). Motor deficit and impairment of synaptic plasticity inmice lacking mGluR1. Nature 372, 237-43.

Cull-Candy, S. G., Brickley, S. G., Misra, C., Feldmeyer, D., Momiyama,A., and Farrant, M. (1998). NMDA receptor diversity in the cerebellum:identification of subunits contributing to functional receptors.Neuropharmacology 37, 1369-80.

Daniels, R. H., Zenke, F. T., and Bokoch, G. M. (1999). alphaPixstimulates p21-activated kinase activity through exchangefactor-dependent and -independent mechanisms. J Biol Chem 274, 6047-50.

Dong, H., O'Brien, R. J., Fung, E. T., Lanahan, A. A., Worley, P. F.,and Huganir, R. L. (1997). GRIP: a synaptic PDZ domain-containingprotein that interacts with AMPA receptors [see comments]. Nature 386,279-84.

DuBois, R. N., and Smalley, W. E. (1996). Cyclooxygenase, NSAIDs, andcolorectal cancer. J Gastroenterol 31, 898-906.

Dudek, S. M., and Bear, M. F. (1989). A biochemical correlate of thecritical period for synaptic modification in the visual cortex. Science246, 673-5.

Dunah, A. W., Luo, J., Wang, Y. H., Yasuda, R. P., and Wolfe, B. B.(1998). Subunit composition of N-methyl-D-aspartate receptors in thecentral nervous system that contain the NR2D subunit. Mol Pharmacol 53,429-37.

Eck, M. J., Dhe-Paganon, S., Trub, T., Nolte, R. T., and Shoelson, S. E.(1996). Structure of the IRS-1 PTB domain bound to the juxtamembraneregion of the insulin receptor. Cell 85, 695-705.

Elmer, G. I., Gorelick, D. A., Goldberg, S. R., and Rothman, R. B.(1996). Acute sensitivity vs. context-specific sensitization to cocaineas a function of genotype. Pharmacol Biochem Behav 53, 623-8.

Emptage, N., Bliss, T. V., and Fine, A. (1999). Single synaptic eventsevoke NMDA receptor-mediated release of calcium from internal stores inhippocampal dendritic spines [In Process Citation]. Neuron 22, 115-24.

Fields, S., and Song, O. (1989). A novel genetic system to detectprotein-protein interactions. Nature 340, 245-6.

Fiorillo, C. D., and Williams, J. T. (1998). Glutamate mediates aninhibitory postsynaptic potential in dopamine neurons [In ProcessCitation]. Nature 394, 78-82.

Fitzgerald, L. W., Ortiz, J., Hamedani, A. G., and Nestler, E. J.(1996). Drugs of abuse and stress increase the expression of GluR1 andNMDAR1 glutamate receptor subunits in the rat ventral tegmental area:common adaptations among cross-sensitizing agents. J Neurosci 16,274-82.

Fosnaugh, J. S., Bhat, R. V., Yamagata, K., Worley, P. F., and Baraban,J. M. (1995). Activation of Arc, a putative “effector” immediate earlygene, by cocaine in rat brain. J. Neurochemistry 64, 2377-2380.

Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P.(1996). Mena, a relative of VASP and Drosophila Enabled, is implicatedin the control of microfilament dynamics. Cell 87, 227-39.

Goebel, D. J., and Poosch, M. S. (1999). NMDA receptor subunit geneexpression in the rat brain: a quantitative analysis of endogenous mRNAlevels of NR1 Com, NR2A, NR2B, NR2C, NR2D and NR3A [In ProcessCitation]. Brain Res Mol Brain Res 69, 164-70.

Golshani, P., Warren, R. A., and Jones, E. G. (1998). Progression ofchange in NMDA, non-NMDA, and metabotropic glutamate receptor functionat the developing corticothalamic synapse. J Neurophysiol 80, 143-54.

Haffner, C., Jarchau, T., Reinhard, M., Hoppe, J., Lohmann, S. M., andWalter, U. (1995). Molecular cloning, structural analysis and functionalexpression of the proline-rich focal adhesion andmicrofilament-associated protein VASP. Embo J 14, 19-27.

Harris, K. M., and Stevens, J. K. (1989). Dendritic spines of CA 1pyramidal cells in the rat hippocampus: serial electron microscopy withreference to their biophysical characteristics. J Neurosci 9, 2982-97.

Harris, K. M., and Stevens, J. K. (1988). Dendritic spines of ratcerebellar Purkinje cells: serial electron microscopy with reference totheir biophysical characteristics. J Neurosci 8,4455-69.

Henry, D. J., and White, F. J. (1991). Repeated cocaine administrationcauses persistent enhancement of D1 dopamine receptor sensitivity withinthe rat nucleus accumbens. J. Pharmacol. Exp. Ther. 258, 882-890.

Herschman, H. R. (1994). Regulation of prostaglandin synthase-1 andprostaglandin synthase-2. Cancer and Metastasis Reviews 13, 241-256.

Hope, B. T., Kosofsky, B., Hyman, S. E., and Nestler, E. J. (1992).Regulation of IEG expression and AP-1 binding by chronic cocaine in therat nucleus accumbens. Proc. Natl. Acad. Sci. USA 89, 5764-5768.

Hsueh, Y. P., and Sheng, M. (1998). Anchoring of glutamate receptors atthe synapse. Prog Brain Res 116, 123-31.

Hyman, S. E. (1996). Addiction to cocaine and amphetamine. Neuron 16,901-4.

Hyvönen, M., Macias, M. J., Nilges, M., Oschkinat, H., Sarastre, M., andWilmanns, M. (1995). Structure of the binding site for inositolphosphates in a PH domain. EMBO J. 14, 4676-4685.

Ikeda, S. R., Lovinger, D. M., McCool, B. A., and Lewis, D. L. (1995).Heterologous expression of metabotropic glutamate receptors in adult ratsympathetic neurons: subtype-specific coupling to ion channels. Neuron14, 1029-38.

Ingi, T., Krumins, A. M., Chidiac, P., Brothers, G. M., Chung, S., Snow,B. E., Barnes, C. A., Lanahan, A. A., Siderovski, D. P., Ross, E. M.,Gilman, A. G., and Worley, P. F. (1998). Dynamic Regulation of RGS2Suggests a Novel Mechanism in G-Protein Signaling and NeuronalPlasticity. J. Neurosci. 18, 7178-7188.

Kammermeier, P., Xiao, B., Tu, J., Worley, P., and Ikeda, S.(submitted). A functional role for the interaction of Homer proteinswith group 1 mGluRs. J. Neuroscience.

Kaufmann, W. E., Worley, P. F., Pegg, J., Bremer, M., and Isakson, P.(1996). COX-2, a synaptically induced enzyme, is expressed by excitatoryneurons at postsynaptic sites in rat cerebral cortex. Proc Natl Acad SciU S A 93, 2317-21.

Kim, E., Naisbitt, S., Hsueh, Y. P., Rao, A., Rothschild, A., Craig, A.M., and Sheng, M. (1997). GKAP, a novel synaptic protein that interactswith the guanylate kinase-like domain of the PSD-95/SAP90 family ofchannel clustering molecules. J Cell Biol 136, 669-78.

Kim, J. H., and Vezina, P. (1998). Metabotropic glutamate receptors arenecessary for sensitization by amphetamine. Neuroreport 9, 403-6.

Koff, J. M., Shuster, L., and Miller, L. G. (1994). Chronic cocaineadministration is associated with behavioral sensitization andtime-dependent changes in striatal dopamine transporter binding. JPharmacol Exp Ther 268, 277-82.

Kombian, S. B., and Malenka, R. C. (1994). Simultaneous LTP of non-NMDA-and LTD of NMDA-receptor-mediated responses in the nucleus accumbens.Nature 368, 242-6.

Kornau, H. C., Schenker, L. T., Kennedy, M. B., and Seeburg, P. H.(1995). Domain interaction between NMDA receptor subunits and thepostsynaptic density protein PSD-95. Science 269, 1737-40.

Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed andschematic plots of protein structures. J. Appl. Cryst. 24, 946-950

Lanahan, A., Lyford, G., Stevenson, G. S., Worley, P. F., and Barnes, C.A. (1997). Selective alteration of long-term potentiation-inducedtranscriptional response in hippocampus of aged, memory-impaired rats.Journal of Neuroscience 1 7, 2876-2885.

Lanahan, A., and Worley, P. (1998). Immediate-Early Genes and SynapticFunction. Neurobiol Learn Mem 70, 37-43.

Lau, L. F., and Nathans, D. (1991). Genes induced by serum growthfactors. In The Hormonal Control of Gene Transcription. Vol. 6.Molecular Aspects, P. Cohen and J. G. Foulkes, eds. (Amsterdam: ElsevierScience Publishers B.V.), pp. 257-293.

Le Moal, M. (1995). Mesocorticolimbic Dopaminergic Neurons; Functionaland Regulatory Roles. In Psychopharmacology: The Fourth Generation ofProgress, F. B. a. D. Kupfer, ed. (New York: Raven Press Ltd.), pp.283-294.

Leanna, C., and Hannink, M. (1996). The reverse two-hybrid system: agenetic scheme for selection against specific protein/proteininteractions. Nucleic Acids Res. 24, 3341-3347.

Leong, P., and MacLennan, D. H. (1998). Complex interactions betweenskeletal muscle ryanodine receptor and dihydropyridine receptor proteins[In Process Citation]. Biochem Cell Biol 76, 681-94.

Leong, P., and MacLennan, D. H. (1998). The cytoplasmic loops betweendomains II and III and domains III and IV in the skeletal muscledihydropyridine receptor bind to a contiguous site in the skeletalmuscle ryanodine receptor. J Biol Chem 273, 29958-64.

Lester, L. B., and Scott, J. D. (1997). Anchoring and scaffold proteinsfor kinases and phosphatases. Recent Prog Horm Res 52, 409-29;discussion 429-30.

Linden, D. J. (1999). The return of the spike: postsynaptic actionpotentials and the induction of LTP and LTD. Neuron 22, 661-6.

Lu, Y. M., Jia, Z., Janus, C., Henderson, J. T., Gerlai, R., Wojtowicz,J. M., and Roder, J. C. (1997). Mice lacking metabotropic glutamatereceptor 5 show impaired learning and reduced CA1 long-term potentiation(LTP) but normal CA3 LTP. J Neurosci 17, 5196-205.

Lujan, R., Nusser, Z., Roberts, J. D., Shigemoto, R., and Somogyi, P.(1996). Perisynaptic location of metabotropic glutamate receptors mGluR1and mGluR5 on dendrites and dendritic spines in the rat hippocampus. EurJ Neurosci 8, 1488-500.

Lujan, R., Roberts, J. D., Shigemoto, R., Ohishi, H., and Somogyi, P.(1997). Differential plasma membrane distribution of metabotropicglutamate receptors mGluR1 alpha, mGluR2 and mGluR5, relative toneurotransmitter release sites. J Chem Neuroanat 13, 219-41.

Lyford, G., Yamagata, K., Kaufmann, W. E., Barnes, C. A., Sanders, L.K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Lanahan, A. A., andWorley, P. F. (1995). Arc, a growth factor and activity-regulated geneencodes a novel cytoskeleton-associated protein that is enriched inneuronal dendrites. Neuron 14, 433-445.

Lyford, G. L., Yamagata, K., Kaufmann, W. E., Barnes, C. A., Sanders, L.K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Lanahan, A. A., andWorley, P. F. (1995). Arc, a growth factor and activity-regulated gene,encodes a novel cytoskeleton-associated protein that is enriched inneuronal dendrites. Neuron 14, 433-45.

Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L.,Tan, I., Leung, T., and Lim, L. (1998). PAK kinases are directly coupledto the PIX family of nucleotide exchange factors. Mol Cell 1, 183-92.

Martin, L. J., Blackstone, C. D., Huganir, R. L., and Price, D. L.(1992). Cellular localization of a metabotropic glutamate receptor inrat brain. Neuron 9, 259-70.

Marx, S. O., Ondrias, K., and Marks, A. R. (1998). Coupled gatingbetween individual skeletal muscle Ca2+ release channels (Ryanodinereceptors) [In Process Citation]. Science 281, 818-21.

Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K.,Tsukita, S., and Tsukita, S. (1998). Rho-kinase phosphorylatesCOOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins andregulates their head-to-tail association. J Cell Biol 140, 647-57.

Miserendino, M., Guitart, X., Terwilliger, R., Chi, S., and Nestler, E.J. (1993). Individual differences in locomotor activity are associatedwith levels of tyrosine hydroxylase and neurofilament proteins in theventral tegmental area of Sprague Dawley rats. Mol. Cell. Neurosci. 4,440-448.

Moratalla, R., Elibol, B., Vallejo, M., and Graybiel, A. M. (1996).Network-level changes in expression of inducible Fos-Jun proteins in thestriatum during chronic cocaine treatment and withdrawal. Neuron 17,147-56.

Morgan, J. I., and Curran, T. (1991). Stimulus-transcription coupling inthe nervous system. Ann. Rev. Neurosci. 14, 421-452.

Naisbitt, S., Kim, E., Tu, J., Xiao, B., Sala, C., Valtschanoff, J.,Weinberg, R., Worley, P., and Sheng, M. (1999). Shank, a novel family ofpostsynaptic density proteins that binds to the NMDAreceptor/PSD-95/GKAP complex and cortactin. Neuron 23, 569-582.

Naisbitt, S., Kim, E., Weinberg, R. J., Rao, A., Yang, F. C., Craig, A.M., and Sheng, M. (1997). Characterization of guanylatekinase-associated protein, a postsynaptic density protein at excitatorysynapses that interacts directly with postsynapticdensity-95/synapse-associated protein 90. J Neurosci 17, 5687-96.

Nakanishi, S., Masu, M., Bessho, Y., Nakajima, Y., Hayashi, Y., andShigemoto, R. (1994). Molecular diversity of glutamate receptors andtheir physiological functions. [Review]. Exs 71, 71-80.

Narasimhan, K., Pessah, I. N., and Linden, D. J. (1998).Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is notrequired for cerebellar long-term depression in reduced preparations. JNeurophysiol 80, 2963-74.

Nestler, E. J., and Aghajanian, G. K. (1997). Molecular and cellularbasis of addiction. Science 278, 58-63.

Nestler, E. J., Hope, B. T., and Widnell, K. L. (1993). Drug addiction:a model for the molecular basis of neural plasticity. Neuron 11,995-1006.

Nestler, E. J., Terwilliger, R. Z., Walker, J. R., Servarino, K. A., andDuman, R. S. (1990). Chronic cocaine treatment decreases levels of theG-protein subunits Gia and Goa in discrete regions of rat brain. J.Neurochem. 55, 1079-1082.

Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D.,Walter, U., Gertler, F. B., Wehland, J., and Chakraborty, T. (1997). Anovel proline-rich motif present in ActA of Listeria monocytogenes andcytoskeletal proteins is the ligand for the EVH1 domain, a proteinmodule present in the Ena/VASP family. EMBO J 16, 5433-44.

Nusser, Z., Mulvihill, E., Streit, P., and Somogyi, P. (1994).Subsynaptic segregation of metabotropic and ionotropic glutamatereceptors as revealed by immunogold localization. Neuroscience 61,421-7.

O'Brien, R. J., Xu, D., Petralia, R. S., Steward, O., Huganir, R. L.,and Worley, P. F. (1999). Synaptic clustering of AMPA receptors by theextracellular immediate-early gene product Narp. Neuron 23, 309-323.

O'Rourke, B., Kass, D. A., Tomaselli, G. F., Kaab, S., Tunin, R., andMarban, E. (1999). Mechanisms of altered excitation-contraction couplingin canine tachycardia-induced heart failure, I: experimental studies.Circ Res 84, 562-70.

Okabe, S., Collin, C., Auerbach, J. M., Meiri, N., Bengzon, J., Kennedy,M. B., Segal, M., and McKay, R. D. (1998). Hippocampal synapticplasticity in mice overexpressing an embryonic subunit of the NMDAreceptor. J Neurosci 18, 4177-88.

Otani, S., and Connor, J. A. (1998). Requirement of rapid Ca2+ entry andsynaptic activation of metabotropic glutamate receptors for theinduction of long-term depression in adult rat hippocampus. J Physiol(Lond) 511, 761-70.

Petralia, R., Esteban, J., Wang, Y.-X., Partridge, J., Zhao, H.-M.,Wenthold, R., and Malinow, R. (1999). Selective acquisition of AMPAreceptors over postnatal development suggests a molecular basis forsilent synapses. Nature Neurosci. 2, 31-36.

Philipp, S., and Flockerzi, V. (1997). Molecular characterization of anovel human PDZ domain protein with homology to INAD from Drosophilamelanogaster. FEBS Lett 413, 243-8.

Piazza, P. V., Deminiere, J.-M., LeMoal, M., and Simon, H. (1989).Factors that predict individual vulnerability to amphetamineself-administration. Science 245, 1511-1513.

Pin, J. P., and Duvoisin, R. (1995). The metabotropic glutamatereceptors: structure and functions. [Review]. Neuropharmacology 34,1-26.

Pisabarro, M. T., Serrano, L., and Wilmanns, M. (1998). CrystalStructure of the Ab1-SH3 Domain Complexed with a Designed High- affinityPeptide Ligand: Implications for SH3-Ligand Interactions. J Mol Biol281, 513-21.

Ponting, C. P., and Phillips, C. (1997). Identification of homer as ahomologue of the Wiskott-Aldrich syndrome protein suggests areceptor-binding function for WH1 domains. J Mol Med 75, 769-71.

Prehoda, K. E., Lee, D. J., and Lim, W. A. (1999). Strucute of theEnabled/VASP Homology 1 domain-peptide complex: A key component in thespatial control of actin assembly. Cell 97, 471-480.

Qian, Z., Gilbert, M. E., Colicos, M. A., Kandel, E. R., and Kuhl, D.(1993). Tissue-plasminogen activator is induced as an immediate-earlygene during seizure, kindling and long-term potentiation. Nature 361,453-457.

Riedel, G. (1996). Function of metabotropic glutamate receptors inlearning and memory. Trends in Neurosciences 19, 219-224.

Robinson, T. E., and Kolb, B. (1999). Alterations in the morphology ofdendrites and dendritic spines in the nucleus accumbens and prefrontalcortex following repeated treatment with amphetamine or cocaine. Eur JNeurosci 11, 1598-604.

Roche, K., Tu, J., Petralia, R., Xiao, B., Wenthold, R., and Worley, P.(accepted). Homer 1b regulates the surface expression of type Imetabotropic glutamate receptors. J. Biol. Chem.

Romano, C., Sesma, M. A., McDonald, C. T., O'Malley, K., Van den Pol, A.N., and Olney, J. W. (1995). Distribution of metabotropic glutamatereceptor mGluR5 immunoreactivity in rat brain. J Comp Neurol 355,455-69.

Ross, C. A., Meldolesi, J., Milner, T. A., Satoh, T., Supattapone, S.,and Snyder, S. H. (1989). Inositol 1,4,5-trisphosphate receptorlocalized to endoplasmic reticulum in cerebellar Purkinje neurons.Nature 339, 468-70.

Saffen, D. W., Cole, A. J., Worley, P. F., Christy, B. A., Ryder, K.,and Baraban, J. M. (1988). Convulsant-induced increase in transcriptionfactor messenger RNAs in rat brain. Proceedings of the National Academyof Sciences (USA) 85, 7795-7799.

Saiki, Y., El-Hayek, R., and Ikemoto, N. (1999). Involvement of theGlu724-Pro760 region of the dihydropyridine receptor II-III loop inskeletal muscle-type excitation-contraction coupling. J Biol Chem 274,7825-32.

Satoh, T., Ross, C. A., Villa, A., Supattapone, S., Pozzan, T., Snyder,S. H., and Meldolesi, J. (1990). The inositol 1,4,5,-trisphosphatereceptor in cerebellar Purkinje cells: quantitative immunogold labelingreveals concentration in an ER subcompartment. J Cell Biol 111 , 615-24.

Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J., Perkins,W., Lee, L., and Isakson, P. (1994). Pharmacological and biochemicaldemonstration of the role of cyclooxygenase 2 in inflammation and pain.Proc Natl Acad Sci U S A 91, 12013-7.

Selig, D. K., Lee, H. K., Bear, M. F., and Malenka, R. C. (1995).Reexamination of the effects of MCPG on hippocampal LTP, LTD, anddepotentiation. J Neurophysiol 74, 1075-82.

Shatz, C. J. (1990). Impulse activity and the patterning of connectionsduring CNS development. Neuron 5, 745-756.

Shaw, R. J., Henry, M., Solomon, F., and Jacks, T. (1998).RhoA-dependent phosphorylation and relocalization of ERM proteins intoapical membrane/actin protrusions in fibroblasts. Mol Biol Cell 9,403-19.

Sheng, M., and Wyszynski, M. (1997). Ion channel targeting in neurons.Bioessays 19, 847-53.

Shih, H., Goldman, P., DeMaggio, A., Hollenberg, S., Goodman, R., andHoekstra, M. (1996). A positive genetic selection for disruptingprotein-protein interactions:Identification of CREB mutations thatprevent association with the coactivator CBP. Proc. Natl. Acad. Sci. USA93, 13896-13901.

Spacek, J., and Harris, K. M. (1997). Three-dimensional organization ofsmooth endoplasmic reticulum in hippocampal CA1 dendrites and dendriticspines of the immature and mature rat. J Neurosci 17, 190-203.

Steward, O., Wallace, C. S., Lyford, G. L., and Worley, P. F. (1998).Synaptic activation causes the mRNA for the immediate early gene Arc tolocalize selectively near activated postsynaptic sites on neuronaldendrites. Neuron 21, 741-751.

Stewart, W. F., Kawas, C., Corrada, M., and Metter, E. J. (1997). Riskof Alzheimer's disease and duration of NSAID use [see comments].Neurology 48, 626-32.

Storck, T., Kruth, U., Kolhekar, R., Sprengel, R., and Seeburg, P. H.(1996). Rapid construction in yeast of complex targeting vectors forgene manipulation in the mouse. Nucleic Acids Res 24, 4594-6.

Svoboda, K., and Mainen, Z. F. (1999). Synaptic [Ca2+]: intracellularstores spill their guts. Neuron 22, 427-30.

Symons, M., Derry, J. M., Karlak, B., Jiang, S., Lemahieu, V.,McCormick, F., Francke, U., and Abo, A. (1996). Wiskott-Aldrich syndromeprotein, a novel effector for the GTPase CDC42Hs, is implicated in actinpolymerization. Cell 84, 723-34.

Taber, M. T., and Fibiger, H. C. (1995). Electrical stimulation of theprefrontal cortex increases dopamine release in the nucleus accumbens ofthe rat: modulation by metabotropic glutamate receptors. J Neurosci 15,3896-904.

Takei, K., Mignery, G. A., Mugnaini, E., Sudhof, T. C., and De Camilli,P. (1994). Inositol 1,4,5-trisphosphate receptor causes formation of ERcisternal stacks in transfected fibroblasts and in cerebellar Purkinjecells. Neuron 12, 327-42.

Takeuchi, M., Hata, Y., Hirao, K., Toyoda, A., Irie, M., and Takai, Y.(1997). SAPAPs. A family of PSD-95/SAP90-associated proteins localizedat postsynaptic density. J Biol Chem 272, 11943-51.

Testa, C. M., Standaert, D. G., Landwehrmeyer, G. B., Penney, J., Jr.,and Young, A. B. (1995). Differential expression of mGluR5 metabotropicglutamate receptor mRNA by rat striatal neurons. J Comp Neurol 354,241-52.

Tolliver, B. K., and Carney, J. M. (1994). Comparison of cocaine and GBR12935: effects on locomotor activity and stereotypy in two inbred mousestrains. Pharmacol Biochem Behav 48, 733-9.

Tsui, C. C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Barnes,C., and Worley, P. F. (1996). Narp, a novel member of the pentraxinfamily, promotes neuite outgrowth and is dynamically regulated byneuronal activity. Journal of Neuroscience 16, 2463-2478.

Tsunoda, S., Sierralta, J., Sun, Y., Bodner, R., Suzuki, E., Becker, A.,Socolich, M., and Zuker, C. S. (1997). A multivalent PDZ-domain proteinassembles signalling complexes in a G- protein-coupled cascade. Nature388, 243-9.

Tu, J. C., Bo Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S.,Brakeman, P. R., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley,P. (1999). mGluR/Homer and PSD-95 Complexes Are Linked by the ShankFamily of Postsynaptic Density Proteins. Neuron 23, 583-592.

Tu, J. C., Xiao, B., Yuan, J., Lanahan, A., Leoffert, K., Li, M.,Linden, D., and Worley, P. F. (1998). Homer binds a novel proline richmotif and links group1 metabotropic glutamate receptors with IP3receptors. Neuron 21, 717-726.

Ujike, H., Okumura, K., Zushi, Y., Akiyama, K., and Otsuki, S. (1992).Persistent supersensitivity of sigma receptors develops during repeatedmethamphetamine treatment. Eur J Pharmacol 211, 323-8.

Vicini, S., Wang, J. F., Li, J. H., Zhu, W. J., Wang, Y. H., Luo, J. H.,Wolfe, B. B., and Grayson, D. R. (1998). Functional and pharmacologicaldifferences between recombinant N-methyl-D-aspartate receptors. JNeurophysiol 79, 555-66.

Vidal, M., Brachmann, R., Fattaey, A., Harlow, E., and Boeke, J. (1996).Reverse two-hybrid and one-hybrid systems to detect dissociation ofprotein-protein and DNA-protein interactions. Proc. Natl. Acad. Sci. USA93, 10315-10320.

Vidal, M., Pascal, B., Chen, E., Boeke, J., and Harlow, E. (1996).Genetic characterization of a mammalian protein-protein interactiondomain by using a yeast reverse two-hybrid system. Proc. Natl. Acad.Sci. USA 93, 10321-10326.

Villa, A., Sharp, A. H., Racchetti, G., Podini, P., Bole, D. G., Dunn,W. A., Pozzan, T., Snyder, S. H., and Meldolesi, J. (1992). Theendoplasmic reticulum of Purkinje neuron body and dendrites: molecularidentity and specializations for Ca2+ transport. Neuroscience 49,467-77.

Vrana, S. L., Vrana, K. E., Koves, T. R., Smith, J. E., and Dworkin, S.I. (1993). Chronic cocaine administration increases CNS tryosinehydroxylase enzyme activity and mRNA levels and trypophan hydroxylaseenzyme activity levels. J. Neurochem. 61, 2262-2268.

Wlodawer, A., Hodgson, K. O., and Shooter, E. M. (1975). Crystallizationof nerve growth factor from mouse submaxillary glands. Proc Natl AcadSci U S A 72, 777-9.

Wolf, M. E. (1998). The role of excitatory amino acids in behavioralsensitization to psychomotor stimulants. Prog Neurobiol 54, 679-720.

Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987).Inositol trisphosphate receptor localization in brain: variablestoichiometry with protein kinase C. Nature 325, 159-161.

Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., andSnyder, S. H. (1987). Characterization of inositol trisphosphatereceptor binding in brain: Regulation by calcium and pH. JournalBiological Chemistry 262, 12132-12136.

Worley, P. F., Cole, A. J., Saffen, D. W., and Baraban, J. M. (1990).Transcription factor regulation in brain: focus on activity and NMDAdependent regulation. In Molecular mechanisms of aging, K. Beyreutherand G. Schettler, eds. (Heidelberg: Springer-Verlag), pp. 62-76.

Xiao, B., Tu, J. C., Petralia, R. S., Yuan, J., Doan, A., Breder, C.,Ruggiero, A., Lanahan, A. A., Wenthold, R. J., and Worley, P. F. (1998).Homer regulates the association of Group 1 metabotropic receptors withmultivalent complexes of Homer-related, synaptic proteins. Neuron 21,707-716.

Yamagata, K., Andreasson, K. I., Kaufmann, W. E., Barnes, C. A., andWorley, P. F. (1993). Expression of a Mitogen-Inducible Cyclooxygenasein Brain Neurons: Regulation by Synaptic Activity and Glucocorticoids.Neuron 11, 371-386.

Yamagata, K., Sanders, L. K., Kaufmann, W. E., Barnes, C. A., Nathans,D., and Worley, P. F. (1994). Rheb, a growth factor and synapticactivity regulated gene, encodes a novel Ras-related protein. Journal ofBiological Chemistry 269, 16333-16339.

Yee, W., and Worley, P. F. (1997). Rheb interacts with Raf-1 kinase andmay function to integrate growth factor- and protein kinase A-dependentsignals. Molecular and Cellular Biology 17, 921-933.

Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., andSchreiber, S. L. (1994). Structural basis for the binding ofproline-rich peptides to SH3 domains. Cell 76, 933-45.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

TABLE 1 Data Collection, Phase Calculation, and Refinement StatisticsWavelength (λ) 0.9879 0.9793 0.9790 0.9611 MAD Data CollectionStatistics Unique reflections 24051 24179 24226 24481 Redundancy 6.2 6.26.2 6.3 Completeness (%) 96.8 97.2 97.3 98.4 Signal (<I>/σ<I>)^(a) 21.5(2.3) 21.0 (2.1) 20.9 (2.0) 20.4 (1.9) R_(sym) (%) 8.1 8.7 9.1 8.8Overall figure of merit 0.71 MAD Structure Factor Ratios^(b) andAnomalous Scattering Factors^(c) 0.9879 0.033 0.040 0.032 0.026 0.97930.047 0.029 0.044 0.9790 0.063 0.036 0.9611 0.050 f′ (e) −4.87 −9.96−8.06 −4.15 f″ (e) 0.47 3.77 6.28 4.12 Refinement Statistics R_(crys)t(%) 25.3 R_(free) (%) 28.4 Average B (Å²) 24.8 protein/31.7 solvent No.of water molecules 88 RMSD bond lengths (Å) 0.0126 RMSD bond angles (°)1.745 RMSD B values (Å²) 0.837/1.487 bonds/angles main chain1.0211/1.594 bonds/angles side chains ^(a)Values in parentheses are forthe highest resolution shell (1.73-1.70Å). R_(sym) = 100 × Σ | I − <I>|/ΣI where I is the integrated intensity of a given reflection. ^(b)RMS (Δ|F|) /RMS (|F |) where Δ |F | is the Bijvoet difference at onewavelength (values on the diagonal) or the dispersive differencesbetween two wavelength (values off the diagonal). ^(c)Anomalouscomponents of the Se scattering factors as a function of wavelength asdetermined by SOLVE (Terwilliger and Eisenberg, 1983). ^(d)All rounds ofrefinement included data for which |F |>2.0σ. R value =Σ|F_(p)(obs)−F_(p)(calc) |/ ΣF_(p)(obs), where F_(p) is the structurefactor amplitude. The free R value was calculated from 10% of the datathat was excluded from the refinement (Brünger, 1992).

Amino Acid Residues and the Homer Binding Domain Expression LevelMutation (Western Blot) Binding^(a) Homer 2 EVH WT ++ +− F7A − NDF7R + + S8L + − N23A ++ + S28A + − V34M ++ + S35V ++ + D39A ++ − R42E ++− R42A ++ + R46A ++ − R46C ++ + I48A ++ + N58A ++ + N64G ++ + F67S + −K69A ++ + Q72A ++ + F74A ++ + F74L ++ + F90S ++ + E93K + + H95A ++ +L96S + + F109C ++ + ^(a)(−) indicates substantially reduced bindingrelative to wild-type (+).

TABLE 2 WASP EVH1 Mutations WASP Residue/Mutation Homer Residue Table2A - β1 region Exposed L39M Met 1 C43W Pro 5 L46P Ser 8 T481 Arg 10E133K His 95 Buried/partially buried T45M Phe 7 A47D Thr 9 A49E Ala 11Table 2B - β3 region Exposed S82P/F Arg 42 Buried/partially buried F84LVal 44 R86C/H/P/L Arg 46 G89D Ser 49 Table 2C - Other mutations ExposedP58L Pro 18 E131K Glu 93 Buried/partially buried H68P Ser 28 V75M Ser 35Y107S/C Phe 67 G125R Gly 87 F128S Phe 90 A134T/V Leu 96 Other A56V —W97C —

Homer Sequence Listing SEQ ID No. Sequence 1 Human Homer 1a (nucleicacid) 2 Human Homer 1a (amino acid) 3 Human Homer 1b (nucleic acid) 4Human Homer 1b (amino acid) 5 Homer 1c (nucleic acid) 6 Homer 1c (aminoacid) 7 Human Homer 2a (nucleic acid) 8 Human Homer 2a (amino acid) 9Human Homer 2b (nucleic acid) 10 Human Homer 2b (amino acid) 11 HumanHomer 3 (nucleic acid) 12 Human Homer 3 (amino acid) 13 peptidebinding—core region: PPXXFR 14 peptide binding—extended region:ALTPPSPFRD 15 Homer interacting protein: rat I30 (nucleic acid) 16 Homerinteracting protein: rat I30 (arnino acid) 17 Homer interacting protein:rat I42 (nucleic acid) 18 Homer interacting protein: rat I42 (aminoacid) 19 Homer interacting protein: human I30 (nucleic acid) 20 Homerinteracting protein: human I30 (amino acid) 21 Homer interactingprotein: human I42 (nucleie acid) 22 Homer interacting protein: humanI42 (amino acid) 23 Mouse Homer 1 a (nucleic acid) 24 Mouse Homer 1 a(amino acid) 25 Mouse Homer 1b (nucleic acid) 26 Mouse Homer 1b (aminoacid) 27 Mouse Homer 2a (nucleic acid) 28 Mouse Homer 2a (amino acid) 29Mouse Homer 2b (nucleic acid) 30 Mouse Homer 2b (amino acid) 31 MouseHomer 3 (nucleic acid) 32 Mouse Homer 3 (amino acid) 33 Rat Homer 1a(nucleic acid) 34 Rat Homer 1a (amino acid) 35 Rat Homer 1b (nucleicacid) 36 Rat Homer 1b (amino acid) 37 Rat Homer 1c (nucleic acid) 38 RatHomer 1c (amino acid) 39 Rat Shank 3a (nucleic acid) 40 Rat Shank 3a(amino acid) 41 Human Homer 3a (nucleic acid) 42 Human Homer 3a (aminoacid) 43 Rat NADL partial nucleic acid sequence 44 Rat NADL partialamino acid sequence

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 72 <210> SEQ ID NO 1 <211>LENGTH: 1084 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (76)..(789) <400> SEQUENCE: 1agacggagaa attcctttgg aagttattcc gtagcataag agctgaaact tcagagcaag 60ttttcattgg gcaaa atg ggg gaa caa cct atc ttc agc act cga gct cat 111 MetGly Glu Gln Pro Ile Phe Ser Thr Arg Ala His 1 5 10 gtc ttc caa att gaccca aac aca aag aag aac tgg gta ccc acc agc 159 Val Phe Gln Ile Asp ProAsn Thr Lys Lys Asn Trp Val Pro Thr Ser 15 20 25 aag cat gca gtt act gtgtct tat ttc tat gac agc aca aga aat gtg 207 Lys His Ala Val Thr Val SerTyr Phe Tyr Asp Ser Thr Arg Asn Val 30 35 40 tat agg ata atc agt tta gatggc tca aag gca ata ata aat agt acc 255 Tyr Arg Ile Ile Ser Leu Asp GlySer Lys Ala Ile Ile Asn Ser Thr 45 50 55 60 atc acc cca aac atg aca tttact aaa aca tct cag aag ttt ggc cag 303 Ile Thr Pro Asn Met Thr Phe ThrLys Thr Ser Gln Lys Phe Gly Gln 65 70 75 tgg gct gat agc cgg gca aac accgtt tat gga ttg gga ttc tcc tct 351 Trp Ala Asp Ser Arg Ala Asn Thr ValTyr Gly Leu Gly Phe Ser Ser 80 85 90 gag cat cat ctt tcg aaa ttt gca gaaaag ttt cag gaa ttt aaa gaa 399 Glu His His Leu Ser Lys Phe Ala Glu LysPhe Gln Glu Phe Lys Glu 95 100 105 gct gct cga cta gca aag gaa aaa tcacaa gag aag atg gaa ctt acc 447 Ala Ala Arg Leu Ala Lys Glu Lys Ser GlnGlu Lys Met Glu Leu Thr 110 115 120 agt aca cct tca cag gaa tcc gca ggcggg gat ctt cag tct cct tta 495 Ser Thr Pro Ser Gln Glu Ser Ala Gly GlyAsp Leu Gln Ser Pro Leu 125 130 135 140 aca ccg gaa agt atc aac ggg acagat gat gaa aga aca cct gat gtg 543 Thr Pro Glu Ser Ile Asn Gly Thr AspAsp Glu Arg Thr Pro Asp Val 145 150 155 aca cag aac tca gag cca agg gctgaa cca act cag aat gca ttg cca 591 Thr Gln Asn Ser Glu Pro Arg Ala GluPro Thr Gln Asn Ala Leu Pro 160 165 170 ttt tca cat agt tca gca atc agcaaa cat tgg gag gct gaa ctg gct 639 Phe Ser His Ser Ser Ala Ile Ser LysHis Trp Glu Ala Glu Leu Ala 175 180 185 acc ctc aaa gga aat aat gcc aaactc act gca gcc ctg ctg gag tcc 687 Thr Leu Lys Gly Asn Asn Ala Lys LeuThr Ala Ala Leu Leu Glu Ser 190 195 200 act gcc aat gtg aaa caa tgg aaacag caa ctt gct gcc tat caa gag 735 Thr Ala Asn Val Lys Gln Trp Lys GlnGln Leu Ala Ala Tyr Gln Glu 205 210 215 220 gaa gca gaa cgt ctg cac aagcgg gta att tca ggg ctg atg tct ata 783 Glu Ala Glu Arg Leu His Lys ArgVal Ile Ser Gly Leu Met Ser Ile 225 230 235 ggg att tagggctaacaggttttctt gatcagaaga aatttgcatg tagattcagc 839 Gly Ile acagggatatcttctagttc taggatgtca gaacatagat atgggttgta tgatatgcat 899 ttgtttgattaagaaaaata ttttccatag tttaatgaga atgaagaata ataccgcctt 959 ttgaagtcaacaaaccatgt tgattcccca tattatccat ggggactagc agtaatgcac 1019 aagtacataaaagcactaat gtattagtgc tagttgatta gtactgacat ggtagttaaa 1079 gtgga 1084<210> SEQ ID NO 2 <211> LENGTH: 238 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 2 Met Gly Glu Gln Pro Ile Phe Ser Thr Arg AlaHis Val Phe Gln Ile 1 5 10 15 Asp Pro Asn Thr Lys Lys Asn Trp Val ProThr Ser Lys His Ala Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Ser Thr ArgAsn Val Tyr Arg Ile Ile 35 40 45 Ser Leu Asp Gly Ser Lys Ala Ile Ile AsnSer Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln Lys PheGly Gln Trp Ala Asp Ser 65 70 75 80 Arg Ala Asn Thr Val Tyr Gly Leu GlyPhe Ser Ser Glu His His Leu 85 90 95 Ser Lys Phe Ala Glu Lys Phe Gln GluPhe Lys Glu Ala Ala Arg Leu 100 105 110 Ala Lys Glu Lys Ser Gln Glu LysMet Glu Leu Thr Ser Thr Pro Ser 115 120 125 Gln Glu Ser Ala Gly Gly AspLeu Gln Ser Pro Leu Thr Pro Glu Ser 130 135 140 Ile Asn Gly Thr Asp AspGlu Arg Thr Pro Asp Val Thr Gln Asn Ser 145 150 155 160 Glu Pro Arg AlaGlu Pro Thr Gln Asn Ala Leu Pro Phe Ser His Ser 165 170 175 Ser Ala IleSer Lys His Trp Glu Ala Glu Leu Ala Thr Leu Lys Gly 180 185 190 Asn AsnAla Lys Leu Thr Ala Ala Leu Leu Glu Ser Thr Ala Asn Val 195 200 205 LysGln Trp Lys Gln Gln Leu Ala Ala Tyr Gln Glu Glu Ala Glu Arg 210 215 220Leu His Lys Arg Val Ile Ser Gly Leu Met Ser Ile Gly Ile 225 230 235<210> SEQ ID NO 3 <211> LENGTH: 1166 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1062) <400> SEQUENCE: 3 atg ggg gag cag ccg att ttc agc act cgagct cat gtc ttc caa att 48 Met Gly Glu Gln Pro Ile Phe Ser Thr Arg AlaHis Val Phe Gln Ile 1 5 10 15 gac cca aac aca aag aag aac tgg gta cccacc agc aag cat gca gtt 96 Asp Pro Asn Thr Lys Lys Asn Trp Val Pro ThrSer Lys His Ala Val 20 25 30 act gtg tct tat ttc tat gac agc aca aga aatgtg tat agg ata atc 144 Thr Val Ser Tyr Phe Tyr Asp Ser Thr Arg Asn ValTyr Arg Ile Ile 35 40 45 agt tta gat ggc tca aag gca ata ata aat agt accatc acc cca aac 192 Ser Leu Asp Gly Ser Lys Ala Ile Ile Asn Ser Thr IleThr Pro Asn 50 55 60 atg aca ttt act aaa aca tct cag aag ttt ggc cag tgggct gat agc 240 Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp AlaAsp Ser 65 70 75 80 cgg gca aac acc gtt tat gga ttg gga ttc tcc tct gagcat cat ctt 288 Arg Ala Asn Thr Val Tyr Gly Leu Gly Phe Ser Ser Glu HisHis Leu 85 90 95 tcg aaa ttt gca gaa aag ttt cag gaa ttt aaa gaa gct gctcga cta 336 Ser Lys Phe Ala Glu Lys Phe Gln Glu Phe Lys Glu Ala Ala ArgLeu 100 105 110 gca aag gaa aaa tca caa gag aag atg gaa ctt acc agt acacct tca 384 Ala Lys Glu Lys Ser Gln Glu Lys Met Glu Leu Thr Ser Thr ProSer 115 120 125 cag gaa tcc gca ggc ggg gat ctt cag tct cct tta aca ccggaa agt 432 Gln Glu Ser Ala Gly Gly Asp Leu Gln Ser Pro Leu Thr Pro GluSer 130 135 140 atc aac ggg aca gat gat gaa aga aca cct gat gtg aca cagaac tca 480 Ile Asn Gly Thr Asp Asp Glu Arg Thr Pro Asp Val Thr Gln AsnSer 145 150 155 160 gag cca agg gct gaa cca act cag aat gca ttg cca ttttca cat agt 528 Glu Pro Arg Ala Glu Pro Thr Gln Asn Ala Leu Pro Phe SerHis Ser 165 170 175 tca gca atc agc aaa cat tgg gag gct gaa ctg gct accctc aaa gga 576 Ser Ala Ile Ser Lys His Trp Glu Ala Glu Leu Ala Thr LeuLys Gly 180 185 190 aat aat gcc aaa ctc act gca gcc ctg ctg gag tcc actgcc aat gtg 624 Asn Asn Ala Lys Leu Thr Ala Ala Leu Leu Glu Ser Thr AlaAsn Val 195 200 205 aaa caa tgg aaa cag caa ctt gct gcc tat caa gag gaagca gaa cgt 672 Lys Gln Trp Lys Gln Gln Leu Ala Ala Tyr Gln Glu Glu AlaGlu Arg 210 215 220 ctg cac aag cgg gtg act gaa ctt gaa tgt gtt agt agccaa gca aat 720 Leu His Lys Arg Val Thr Glu Leu Glu Cys Val Ser Ser GlnAla Asn 225 230 235 240 gca gta cat act cat aag aca gaa tta aat cag acaata caa gaa ctg 768 Ala Val His Thr His Lys Thr Glu Leu Asn Gln Thr IleGln Glu Leu 245 250 255 gaa gag aca ctg aaa ctg aag gaa gag gaa ata gaaagg tta aaa caa 816 Glu Glu Thr Leu Lys Leu Lys Glu Glu Glu Ile Glu ArgLeu Lys Gln 260 265 270 gaa att gat aat gcc aga gaa cta caa gaa cag agggat tct ttg act 864 Glu Ile Asp Asn Ala Arg Glu Leu Gln Glu Gln Arg AspSer Leu Thr 275 280 285 cag aaa cta cag gaa gta gaa att cgg aac aaa gacctg gag gga caa 912 Gln Lys Leu Gln Glu Val Glu Ile Arg Asn Lys Asp LeuGlu Gly Gln 290 295 300 ctg tct gac tta gag caa cgt ctg gag aaa agt cagaat gaa caa gaa 960 Leu Ser Asp Leu Glu Gln Arg Leu Glu Lys Ser Gln AsnGlu Gln Glu 305 310 315 320 gct ttt cgc aat aac ctg aag aca ctc tta gaaatt ctg gat gga aag 1008 Ala Phe Arg Asn Asn Leu Lys Thr Leu Leu Glu IleLeu Asp Gly Lys 325 330 335 ata ttt gaa cta aca gaa tta cga gat aac ttggcc aag cta cta gaa 1056 Ile Phe Glu Leu Thr Glu Leu Arg Asp Asn Leu AlaLys Leu Leu Glu 340 345 350 tgc agc taaggaaagt gaaatttcag tgccaattaattaaaagata cactgtctct 1112 Cys Ser cttcatagga ctgtttagct ctgcatcaagattgcacaaa aaaaaaaaaa aaaa 1166 <210> SEQ ID NO 4 <211> LENGTH: 354<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Met GlyGlu Gln Pro Ile Phe Ser Thr Arg Ala His Val Phe Gln Ile 1 5 10 15 AspPro Asn Thr Lys Lys Asn Trp Val Pro Thr Ser Lys His Ala Val 20 25 30 ThrVal Ser Tyr Phe Tyr Asp Ser Thr Arg Asn Val Tyr Arg Ile Ile 35 40 45 SerLeu Asp Gly Ser Lys Ala Ile Ile Asn Ser Thr Ile Thr Pro Asn 50 55 60 MetThr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser 65 70 75 80Arg Ala Asn Thr Val Tyr Gly Leu Gly Phe Ser Ser Glu His His Leu 85 90 95Ser Lys Phe Ala Glu Lys Phe Gln Glu Phe Lys Glu Ala Ala Arg Leu 100 105110 Ala Lys Glu Lys Ser Gln Glu Lys Met Glu Leu Thr Ser Thr Pro Ser 115120 125 Gln Glu Ser Ala Gly Gly Asp Leu Gln Ser Pro Leu Thr Pro Glu Ser130 135 140 Ile Asn Gly Thr Asp Asp Glu Arg Thr Pro Asp Val Thr Gln AsnSer 145 150 155 160 Glu Pro Arg Ala Glu Pro Thr Gln Asn Ala Leu Pro PheSer His Ser 165 170 175 Ser Ala Ile Ser Lys His Trp Glu Ala Glu Leu AlaThr Leu Lys Gly 180 185 190 Asn Asn Ala Lys Leu Thr Ala Ala Leu Leu GluSer Thr Ala Asn Val 195 200 205 Lys Gln Trp Lys Gln Gln Leu Ala Ala TyrGln Glu Glu Ala Glu Arg 210 215 220 Leu His Lys Arg Val Thr Glu Leu GluCys Val Ser Ser Gln Ala Asn 225 230 235 240 Ala Val His Thr His Lys ThrGlu Leu Asn Gln Thr Ile Gln Glu Leu 245 250 255 Glu Glu Thr Leu Lys LeuLys Glu Glu Glu Ile Glu Arg Leu Lys Gln 260 265 270 Glu Ile Asp Asn AlaArg Glu Leu Gln Glu Gln Arg Asp Ser Leu Thr 275 280 285 Gln Lys Leu GlnGlu Val Glu Ile Arg Asn Lys Asp Leu Glu Gly Gln 290 295 300 Leu Ser AspLeu Glu Gln Arg Leu Glu Lys Ser Gln Asn Glu Gln Glu 305 310 315 320 AlaPhe Arg Asn Asn Leu Lys Thr Leu Leu Glu Ile Leu Asp Gly Lys 325 330 335Ile Phe Glu Leu Thr Glu Leu Arg Asp Asn Leu Ala Lys Leu Leu Glu 340 345350 Cys Ser <210> SEQ ID NO 5 <211> LENGTH: 105 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Lys Glu Val Trp Gln Val Ile LeuLys Pro Lys Gly Leu Gly Gln Thr 1 5 10 15 Lys Asn Leu Ile Gly Ile TyrArg Leu Cys Leu Thr Ser Lys Thr Ile 20 25 30 Ser Phe Val Lys Leu Asn SerGlu Ala Ala Ala Val Val Leu Gln Leu 35 40 45 Met Asn Ile Arg Arg Cys GlyHis Ser Glu Asn Phe Phe Phe Ile Glu 50 55 60 Val Gly Arg Ser Ala Val ThrGly Pro Gly Glu Phe Trp Met Gln Val 65 70 75 80 Asp Asp Ser Val Val AlaGln Asn Met His Glu Thr Ile Leu Glu Ala 85 90 95 Met Arg Ala Met Ser AspGlu Phe Arg 100 105 <210> SEQ ID NO 6 <211> LENGTH: 106 <212> TYPE: PRT<213> ORGANISM: Mouse <400> SEQUENCE: 6 Met Glu Gly Phe Leu Asn Arg LysHis Glu Trp Glu Ala His Asn Lys 1 5 10 15 Lys Ala Ser Ser Arg Ser TrpHis Asn Val Tyr Cys Val Ile Asn Asn 20 25 30 Gln Glu Met Gly Phe Tyr LysAsp Ala Lys Ser Ala Ala Ser Gly Ile 35 40 45 Pro Tyr His Ser Glu Val ProVal Ser Leu Lys Glu Ala Ile Cys Glu 50 55 60 Val Ala Leu Asp Tyr Lys LysLys Lys His Val Phe Lys Leu Arg Leu 65 70 75 80 Ser Asp Gly Asn Glu TyrLeu Phe Gln Ala Lys Asp Asp Glu Glu Met 85 90 95 Asn Thr Trp Ile Gln AlaIle Ser Ser Ala 100 105 <210> SEQ ID NO 7 <211> LENGTH: 1767 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(1029) <400> SEQUENCE: 7 atg ggg gag cag ccg atcttc acc acc cga gcg cat gtc ttc cag att 48 Met Gly Glu Gln Pro Ile PheThr Thr Arg Ala His Val Phe Gln Ile 1 5 10 15 gac ccc aac acc aag aagaac tgg atg cct gcg agc aag cag gcg gtc 96 Asp Pro Asn Thr Lys Lys AsnTrp Met Pro Ala Ser Lys Gln Ala Val 20 25 30 acc gtt tcc tac ttc tat gatgtc aca agg aac agc tat cgg atc atc 144 Thr Val Ser Tyr Phe Tyr Asp ValThr Arg Asn Ser Tyr Arg Ile Ile 35 40 45 agt gtg gac gga gcc aag gtg atcata aac agc aca atc aca ccg aat 192 Ser Val Asp Gly Ala Lys Val Ile IleAsn Ser Thr Ile Thr Pro Asn 50 55 60 atg acc ttc acc aaa acg tca cag aagttt ggg cag tgg gcc gac agc 240 Met Thr Phe Thr Lys Thr Ser Gln Lys PheGly Gln Trp Ala Asp Ser 65 70 75 80 aga gcc aac aca gtg ttt ggt ttg gggttt tcc tct gag cag cag ctg 288 Arg Ala Asn Thr Val Phe Gly Leu Gly PheSer Ser Glu Gln Gln Leu 85 90 95 aca aag ttt gca gag aaa ttc cag gag gtgaaa gaa gct gcc aag ata 336 Thr Lys Phe Ala Glu Lys Phe Gln Glu Val LysGlu Ala Ala Lys Ile 100 105 110 gcc aaa gac aag acg cag gag aaa atc gagacc tca agt aat cat tcc 384 Ala Lys Asp Lys Thr Gln Glu Lys Ile Glu ThrSer Ser Asn His Ser 115 120 125 caa gca tcc agt gtc aac ggg acg gac gaggaa aag gcc tct cac gcc 432 Gln Ala Ser Ser Val Asn Gly Thr Asp Glu GluLys Ala Ser His Ala 130 135 140 ggt cca gcc aac aca caa ctg aag tct gagaat gac aag ctg aag att 480 Gly Pro Ala Asn Thr Gln Leu Lys Ser Glu AsnAsp Lys Leu Lys Ile 145 150 155 160 gcc ttg acg cag agc gca gcc aac gtgaag aag tgg gag atc gag ctg 528 Ala Leu Thr Gln Ser Ala Ala Asn Val LysLys Trp Glu Ile Glu Leu 165 170 175 cag acc ctt cgg gag agc aat gca cggctg acc aca gca ctg cag gag 576 Gln Thr Leu Arg Glu Ser Asn Ala Arg LeuThr Thr Ala Leu Gln Glu 180 185 190 tcg gca gcc agt gtg gag cag tgg aagagg cag ttc tcc atc tgc cgt 624 Ser Ala Ala Ser Val Glu Gln Trp Lys ArgGln Phe Ser Ile Cys Arg 195 200 205 gat gag aat gac cgg ctc cgc aac aagatt gat gag ctg gaa gaa caa 672 Asp Glu Asn Asp Arg Leu Arg Asn Lys IleAsp Glu Leu Glu Glu Gln 210 215 220 tgc agt gag atc aac aga gag aag gagaag aac acg cag ctg aag agg 720 Cys Ser Glu Ile Asn Arg Glu Lys Glu LysAsn Thr Gln Leu Lys Arg 225 230 235 240 agg atc gag gag ctg gag gca gagctc cga gaa aag gag aca gag ctg 768 Arg Ile Glu Glu Leu Glu Ala Glu LeuArg Glu Lys Glu Thr Glu Leu 245 250 255 aaa gat ctc cga aaa caa agt gaaatc ata cct cag ctc atg tca gag 816 Lys Asp Leu Arg Lys Gln Ser Glu IleIle Pro Gln Leu Met Ser Glu 260 265 270 tgc gaa tat gtc tct gag aag ctagag gcg gca gag aga gac aat caa 864 Cys Glu Tyr Val Ser Glu Lys Leu GluAla Ala Glu Arg Asp Asn Gln 275 280 285 aac ctg gaa gac aaa gtg cgt tcctta aag aca gac att gag gag agc 912 Asn Leu Glu Asp Lys Val Arg Ser LeuLys Thr Asp Ile Glu Glu Ser 290 295 300 aaa tac cga cag cgc cac ctg aaggtg gag ttg aag agc ttc ctg gag 960 Lys Tyr Arg Gln Arg His Leu Lys ValGlu Leu Lys Ser Phe Leu Glu 305 310 315 320 gtg ctg gac ggg aag att gacgac ctg cat gac ttc cgc cga ggg ctc 1008 Val Leu Asp Gly Lys Ile Asp AspLeu His Asp Phe Arg Arg Gly Leu 325 330 335 tcc aag ctg ggc acc gat aactagggctggc cgaggcccag gccccgcccg 1059 Ser Lys Leu Gly Thr Asp Asn 340tgagtcccaa gcgtgtgtgc gagaccagat agctctagga cgttcttctg tgtgcattgc 1119ttctgtaaat gcaggcgcag tttgtcgtgt ttccaaacca gttgtgccgt ccactcactc 1179cttttcagaa tagaaatctc ctctcgcttc tctggccttg tgaggttgtg gacaactgga 1239agattctgac tcaggaatcc agaactaggt ctaccttcaa catttatgca gtcagggcag 1299ggatgtttat atctttcata agggctgttg caaccatatg aactgaaaaa acacgcattt 1359tgtaatccaa atattgatat tctttacacc aagccatcag gctcctttta tcaaatagca 1419ttcagagtat ttgaatgtcc accagacacc agccccgggg ggcacagaga gaacaacatt 1479cctctctgtc aacatcgaga ggctttaaaa caactgttta gtggaaactt tctgagagat 1539ggaaaacaag cttctggtgg gtgcattttc tggcccggag ttgcctgcat ccacgctact 1599gccccctgcc ccccgccccc ccagtttgta cggttgcaac agtgttcctt ttcttggttt 1659taatttctga gcagatgatt tgctgtggga acagcacaca gtgagggtgc ctagcacaat 1719gtctggcaca aagtaggtgc ttaataaata tttgttcaat taaaaaaa 1767 <210> SEQ IDNO 8 <211> LENGTH: 343 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 8 Met Gly Glu Gln Pro Ile Phe Thr Thr Arg Ala His ValPhe Gln Ile 1 5 10 15 Asp Pro Asn Thr Lys Lys Asn Trp Met Pro Ala SerLys Gln Ala Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Val Thr Arg Asn SerTyr Arg Ile Ile 35 40 45 Ser Val Asp Gly Ala Lys Val Ile Ile Asn Ser ThrIle Thr Pro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly GlnTrp Ala Asp Ser 65 70 75 80 Arg Ala Asn Thr Val Phe Gly Leu Gly Phe SerSer Glu Gln Gln Leu 85 90 95 Thr Lys Phe Ala Glu Lys Phe Gln Glu Val LysGlu Ala Ala Lys Ile 100 105 110 Ala Lys Asp Lys Thr Gln Glu Lys Ile GluThr Ser Ser Asn His Ser 115 120 125 Gln Ala Ser Ser Val Asn Gly Thr AspGlu Glu Lys Ala Ser His Ala 130 135 140 Gly Pro Ala Asn Thr Gln Leu LysSer Glu Asn Asp Lys Leu Lys Ile 145 150 155 160 Ala Leu Thr Gln Ser AlaAla Asn Val Lys Lys Trp Glu Ile Glu Leu 165 170 175 Gln Thr Leu Arg GluSer Asn Ala Arg Leu Thr Thr Ala Leu Gln Glu 180 185 190 Ser Ala Ala SerVal Glu Gln Trp Lys Arg Gln Phe Ser Ile Cys Arg 195 200 205 Asp Glu AsnAsp Arg Leu Arg Asn Lys Ile Asp Glu Leu Glu Glu Gln 210 215 220 Cys SerGlu Ile Asn Arg Glu Lys Glu Lys Asn Thr Gln Leu Lys Arg 225 230 235 240Arg Ile Glu Glu Leu Glu Ala Glu Leu Arg Glu Lys Glu Thr Glu Leu 245 250255 Lys Asp Leu Arg Lys Gln Ser Glu Ile Ile Pro Gln Leu Met Ser Glu 260265 270 Cys Glu Tyr Val Ser Glu Lys Leu Glu Ala Ala Glu Arg Asp Asn Gln275 280 285 Asn Leu Glu Asp Lys Val Arg Ser Leu Lys Thr Asp Ile Glu GluSer 290 295 300 Lys Tyr Arg Gln Arg His Leu Lys Val Glu Leu Lys Ser PheLeu Glu 305 310 315 320 Val Leu Asp Gly Lys Ile Asp Asp Leu His Asp PheArg Arg Gly Leu 325 330 335 Ser Lys Leu Gly Thr Asp Asn 340 <210> SEQ IDNO 9 <211> LENGTH: 1800 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1062) <400>SEQUENCE: 9 atg ggg gag cag ccg atc ttc acc acc cga gcg cat gtc ttc cagatt 48 Met Gly Glu Gln Pro Ile Phe Thr Thr Arg Ala His Val Phe Gln Ile 15 10 15 gac ccc aac acc aag aag aac tgg atg cct gcg agc aag cag gcg gtc96 Asp Pro Asn Thr Lys Lys Asn Trp Met Pro Ala Ser Lys Gln Ala Val 20 2530 acc gtt tcc tac ttc tat gat gtc aca agg aac agc tat cgg atc atc 144Thr Val Ser Tyr Phe Tyr Asp Val Thr Arg Asn Ser Tyr Arg Ile Ile 35 40 45agt gtg gac gga gcc aag gtg atc ata aac agc aca atc aca ccg aat 192 SerVal Asp Gly Ala Lys Val Ile Ile Asn Ser Thr Ile Thr Pro Asn 50 55 60 atgacc ttc acc aaa acg tca cag aag ttt ggg cag tgg gcc gac agc 240 Met ThrPhe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser 65 70 75 80 agagcc aac aca gtg ttt ggt ttg ggg ttt tcc tct gag cag cag ctg 288 Arg AlaAsn Thr Val Phe Gly Leu Gly Phe Ser Ser Glu Gln Gln Leu 85 90 95 aca aagttt gca gag aaa ttc cag gag gtg aaa gaa gct gcc aag ata 336 Thr Lys PheAla Glu Lys Phe Gln Glu Val Lys Glu Ala Ala Lys Ile 100 105 110 gcc aaagac aag acg cag gag aaa atc gag acc tca agt aat cat tcc 384 Ala Lys AspLys Thr Gln Glu Lys Ile Glu Thr Ser Ser Asn His Ser 115 120 125 caa gaatct ggg cgt gaa acc cca tct tct act cag gca tcc agt gtc 432 Gln Glu SerGly Arg Glu Thr Pro Ser Ser Thr Gln Ala Ser Ser Val 130 135 140 aac gggacg gac gag gaa aag gcc tct cac gcc ggt cca gcc aac aca 480 Asn Gly ThrAsp Glu Glu Lys Ala Ser His Ala Gly Pro Ala Asn Thr 145 150 155 160 caactg aag tct gag aat gac aag ctg aag att gcc ttg acg cag agc 528 Gln LeuLys Ser Glu Asn Asp Lys Leu Lys Ile Ala Leu Thr Gln Ser 165 170 175 gcagcc aac gtg aag aag tgg gag atc gag ctg cag acc ctt cgg gag 576 Ala AlaAsn Val Lys Lys Trp Glu Ile Glu Leu Gln Thr Leu Arg Glu 180 185 190 agcaat gca cgg ctg acc aca gca ctg cag gag tcg gca gcc agt gtg 624 Ser AsnAla Arg Leu Thr Thr Ala Leu Gln Glu Ser Ala Ala Ser Val 195 200 205 gagcag tgg aag agg cag ttc tcc atc tgc cgt gat gag aat gac cgg 672 Glu GlnTrp Lys Arg Gln Phe Ser Ile Cys Arg Asp Glu Asn Asp Arg 210 215 220 ctccgc aac aag att gat gag ctg gaa gaa caa tgc agt gag atc aac 720 Leu ArgAsn Lys Ile Asp Glu Leu Glu Glu Gln Cys Ser Glu Ile Asn 225 230 235 240aga gag aag gag aag aac acg cag ctg aag agg agg atc gag gag ctg 768 ArgGlu Lys Glu Lys Asn Thr Gln Leu Lys Arg Arg Ile Glu Glu Leu 245 250 255gag gca gag ctc cga gaa aag gag aca gag ctg aaa gat ctc cga aaa 816 GluAla Glu Leu Arg Glu Lys Glu Thr Glu Leu Lys Asp Leu Arg Lys 260 265 270caa agt gaa atc ata cct cag ctc atg tca gag tgc gaa tat gtc tct 864 GlnSer Glu Ile Ile Pro Gln Leu Met Ser Glu Cys Glu Tyr Val Ser 275 280 285gag aag cta gag gcg gca gag aga gac aat caa aac ctg gaa gac aaa 912 GluLys Leu Glu Ala Ala Glu Arg Asp Asn Gln Asn Leu Glu Asp Lys 290 295 300gtg cgt tcc tta aag aca gac att gag gag agc aaa tac cga cag cgc 960 ValArg Ser Leu Lys Thr Asp Ile Glu Glu Ser Lys Tyr Arg Gln Arg 305 310 315320 cac ctg aag gtg gag ttg aag agc ttc ctg gag gtg ctg gac ggg aag 1008His Leu Lys Val Glu Leu Lys Ser Phe Leu Glu Val Leu Asp Gly Lys 325 330335 att gac gac ctg cat gac ttc cgc cga ggg ctc tcc aag ctg ggc acc 1056Ile Asp Asp Leu His Asp Phe Arg Arg Gly Leu Ser Lys Leu Gly Thr 340 345350 gat aac tagggctggc cgaggcccag gccccgcccg tgagtcccaa gcgtgtgtgc 1112Asp Asn gagaccagat agctctagga cgttcttctg tgtgcattgc ttctgtaaatgcaggcgcag 1172 tttgtcgtgt ttccaaacca gttgtgccgt ccactcactc cttttcagaatagaaatctc 1232 ctctcgcttc tctggccttg tgaggttgtg gacaactgga agattctgactcaggaatcc 1292 agaactaggt ctaccttcaa catttatgca gtcagggcag ggatgtttatatctttcata 1352 agggctgttg caaccatatg aactgaaaaa acacgcattt tgtaatccaaatattgatat 1412 tctttacacc aagccatcag gctcctttta tcaaatagca ttcagagtatttgaatgtcc 1472 accagacacc agccccgggg ggcacagaga gaacaacatt cctctctgtcaacatcgaga 1532 ggctttaaaa caactgttta gtggaaactt tctgagagat ggaaaacaagcttctggtgg 1592 gtgcattttc tggcccggag ttgcctgcat ccacgctact gccccctgccccccgccccc 1652 ccagtttgta cggttgcaac agtgttcctt ttcttggttt taatttctgagcagatgatt 1712 tgctgtggga acagcacaca gtgagggtgc ctagcacaat gtctggcacaaagtaggtgc 1772 ttaataaata tttgttcaat taaaaaaa 1800 <210> SEQ ID NO 10<211> LENGTH: 354 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 10 Met Gly Glu Gln Pro Ile Phe Thr Thr Arg Ala His Val Phe GlnIle 1 5 10 15 Asp Pro Asn Thr Lys Lys Asn Trp Met Pro Ala Ser Lys GlnAla Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Val Thr Arg Asn Ser Tyr ArgIle Ile 35 40 45 Ser Val Asp Gly Ala Lys Val Ile Ile Asn Ser Thr Ile ThrPro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp AlaAsp Ser 65 70 75 80 Arg Ala Asn Thr Val Phe Gly Leu Gly Phe Ser Ser GluGln Gln Leu 85 90 95 Thr Lys Phe Ala Glu Lys Phe Gln Glu Val Lys Glu AlaAla Lys Ile 100 105 110 Ala Lys Asp Lys Thr Gln Glu Lys Ile Glu Thr SerSer Asn His Ser 115 120 125 Gln Glu Ser Gly Arg Glu Thr Pro Ser Ser ThrGln Ala Ser Ser Val 130 135 140 Asn Gly Thr Asp Glu Glu Lys Ala Ser HisAla Gly Pro Ala Asn Thr 145 150 155 160 Gln Leu Lys Ser Glu Asn Asp LysLeu Lys Ile Ala Leu Thr Gln Ser 165 170 175 Ala Ala Asn Val Lys Lys TrpGlu Ile Glu Leu Gln Thr Leu Arg Glu 180 185 190 Ser Asn Ala Arg Leu ThrThr Ala Leu Gln Glu Ser Ala Ala Ser Val 195 200 205 Glu Gln Trp Lys ArgGln Phe Ser Ile Cys Arg Asp Glu Asn Asp Arg 210 215 220 Leu Arg Asn LysIle Asp Glu Leu Glu Glu Gln Cys Ser Glu Ile Asn 225 230 235 240 Arg GluLys Glu Lys Asn Thr Gln Leu Lys Arg Arg Ile Glu Glu Leu 245 250 255 GluAla Glu Leu Arg Glu Lys Glu Thr Glu Leu Lys Asp Leu Arg Lys 260 265 270Gln Ser Glu Ile Ile Pro Gln Leu Met Ser Glu Cys Glu Tyr Val Ser 275 280285 Glu Lys Leu Glu Ala Ala Glu Arg Asp Asn Gln Asn Leu Glu Asp Lys 290295 300 Val Arg Ser Leu Lys Thr Asp Ile Glu Glu Ser Lys Tyr Arg Gln Arg305 310 315 320 His Leu Lys Val Glu Leu Lys Ser Phe Leu Glu Val Leu AspGly Lys 325 330 335 Ile Asp Asp Leu His Asp Phe Arg Arg Gly Leu Ser LysLeu Gly Thr 340 345 350 Asp Asn <210> SEQ ID NO 11 <211> LENGTH: 1429<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(1429) <223> OTHERINFORMATION: n is either a, c, g, or t <221> NAME/KEY: CDS <222>LOCATION: (91)..(1164) <400> SEQUENCE: 11 gcacgagggc gcatgactagttggggccaa accagtgctc ctgccacctc tctggctgcc 60 ccctagagcc tgcccatcccagcctgacca atg tcc aca gcc agg gag cag cca 114 Met Ser Thr Ala Arg GluGln Pro 1 5 atc ttc agc aca cgg gcg cac gtg ttc caa att gac cca gcc accaag 162 Ile Phe Ser Thr Arg Ala His Val Phe Gln Ile Asp Pro Ala Thr Lys10 15 20 cga aac tgg atc cca gcg ggc aag cac gca ctc act gtc tcc tat ttc210 Arg Asn Trp Ile Pro Ala Gly Lys His Ala Leu Thr Val Ser Tyr Phe 2530 35 40 tac gat gcc acc cgc aat gtg tac cgc atc atc agc atc gga ggc gcc258 Tyr Asp Ala Thr Arg Asn Val Tyr Arg Ile Ile Ser Ile Gly Gly Ala 4550 55 aag gcc atc atc aac agc act gtc act ccc aac atg acc ttc acc aaa306 Lys Ala Ile Ile Asn Ser Thr Val Thr Pro Asn Met Thr Phe Thr Lys 6065 70 act tcc cag aag ttc ggg cag tgg gcc gac agt cgc gcc aac aca gtc354 Thr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser Arg Ala Asn Thr Val 7580 85 tac ggc ctg ggc ttt gcc tct gaa cag cat ctg aca cag ttt gcc gag402 Tyr Gly Leu Gly Phe Ala Ser Glu Gln His Leu Thr Gln Phe Ala Glu 9095 100 aag ttc cag gaa gtg aag gaa gca gcc agg ctg gcc agg gag aaa tct450 Lys Phe Gln Glu Val Lys Glu Ala Ala Arg Leu Ala Arg Glu Lys Ser 105110 115 120 cag gat ggc ggg gag ctc acc agt cca gcc ctg ggg ctc gcc tcccac 498 Gln Asp Gly Gly Glu Leu Thr Ser Pro Ala Leu Gly Leu Ala Ser His125 130 135 cag gtc ccc ccg agc cct ctc gtc agt gcc aac ggc ccc ggc gaggaa 546 Gln Val Pro Pro Ser Pro Leu Val Ser Ala Asn Gly Pro Gly Glu Glu140 145 150 aaa ctg ttc cgc agc cag agc gct gat gcc ccc ggc ccc aca gagcgc 594 Lys Leu Phe Arg Ser Gln Ser Ala Asp Ala Pro Gly Pro Thr Glu Arg155 160 165 gag cgg cta aag aag atg ttg tct gag ggc tcc gtg ggc gag gtacag 642 Glu Arg Leu Lys Lys Met Leu Ser Glu Gly Ser Val Gly Glu Val Gln170 175 180 tgg gag gcc gag ttt ttc gca ctg cag gac agc aac aac aag ctggca 690 Trp Glu Ala Glu Phe Phe Ala Leu Gln Asp Ser Asn Asn Lys Leu Ala185 190 195 200 ggc gcc ctg cga gag gcc aac gcc gcc gca gcc cag tgg aggcag cag 738 Gly Ala Leu Arg Glu Ala Asn Ala Ala Ala Ala Gln Trp Arg GlnGln 205 210 215 ctg gag gct cag cgt gca gag gcc gag cgg ctg cgg cag cgggtg gct 786 Leu Glu Ala Gln Arg Ala Glu Ala Glu Arg Leu Arg Gln Arg ValAla 220 225 230 gag ctg gag gct cag gca gct tca gag gtg acc ccc acc ggtgag aag 834 Glu Leu Glu Ala Gln Ala Ala Ser Glu Val Thr Pro Thr Gly GluLys 235 240 245 gag ggg ctg ggc cag ggc cag tcg ctg gaa cag ctg gaa gctctg gtg 882 Glu Gly Leu Gly Gln Gly Gln Ser Leu Glu Gln Leu Glu Ala LeuVal 250 255 260 caa acc aag gac cag gag att cag acc ctg aag agt cag actggg ggg 930 Gln Thr Lys Asp Gln Glu Ile Gln Thr Leu Lys Ser Gln Thr GlyGly 265 270 275 280 ccc cgc gag gcc ctg gag gct gcc gag cgt gag gag actcag cag aag 978 Pro Arg Glu Ala Leu Glu Ala Ala Glu Arg Glu Glu Thr GlnGln Lys 285 290 295 gtg cag acc cgc aat gcg gag ttg gag cac cag ctg cgggcg atg gag 1026 Val Gln Thr Arg Asn Ala Glu Leu Glu His Gln Leu Arg AlaMet Glu 300 305 310 cgc agc ctg gag gag gca cgg gca gag cgg gag cgg gcgcgg gct gag 1074 Arg Ser Leu Glu Glu Ala Arg Ala Glu Arg Glu Arg Ala ArgAla Glu 315 320 325 gtg ggc cgg gca gcg cag ctg ctg gac gtc agc ctg tttgag ctg agt 1122 Val Gly Arg Ala Ala Gln Leu Leu Asp Val Ser Leu Phe GluLeu Ser 330 335 340 gag ctg cgt gag ggc ctg gcc cgc ctg gct gag gct gcgccc 1164 Glu Leu Arg Glu Gly Leu Ala Arg Leu Ala Glu Ala Ala Pro 345 350355 tgagccgggg ctggttttct atgaacgatt ccggcctggg atgcgggcca ggctgcaggc1224 ggcatagttg ggcccattcg tcctggaaag ggactggggg gtcccaactt agccctgggt1284 gggccgggcc gggntgggct ggggtgggcc ccagtcggct ctggttgttg gcagctttgg1344 ggctgttttt gagcttctca ttgtgtagaa tttctagatc ccccgattac atttctaagc1404 gtgaaaaaaa aaaaaaaaaa aaaaa 1429 <210> SEQ ID NO 12 <211> LENGTH:358 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(1429) <223> OTHERINFORMATION: n is either a, c, g, or t <400> SEQUENCE: 12 Met Ser ThrAla Arg Glu Gln Pro Ile Phe Ser Thr Arg Ala His Val 1 5 10 15 Phe GlnIle Asp Pro Ala Thr Lys Arg Asn Trp Ile Pro Ala Gly Lys 20 25 30 His AlaLeu Thr Val Ser Tyr Phe Tyr Asp Ala Thr Arg Asn Val Tyr 35 40 45 Arg IleIle Ser Ile Gly Gly Ala Lys Ala Ile Ile Asn Ser Thr Val 50 55 60 Thr ProAsn Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp 65 70 75 80 AlaAsp Ser Arg Ala Asn Thr Val Tyr Gly Leu Gly Phe Ala Ser Glu 85 90 95 GlnHis Leu Thr Gln Phe Ala Glu Lys Phe Gln Glu Val Lys Glu Ala 100 105 110Ala Arg Leu Ala Arg Glu Lys Ser Gln Asp Gly Gly Glu Leu Thr Ser 115 120125 Pro Ala Leu Gly Leu Ala Ser His Gln Val Pro Pro Ser Pro Leu Val 130135 140 Ser Ala Asn Gly Pro Gly Glu Glu Lys Leu Phe Arg Ser Gln Ser Ala145 150 155 160 Asp Ala Pro Gly Pro Thr Glu Arg Glu Arg Leu Lys Lys MetLeu Ser 165 170 175 Glu Gly Ser Val Gly Glu Val Gln Trp Glu Ala Glu PhePhe Ala Leu 180 185 190 Gln Asp Ser Asn Asn Lys Leu Ala Gly Ala Leu ArgGlu Ala Asn Ala 195 200 205 Ala Ala Ala Gln Trp Arg Gln Gln Leu Glu AlaGln Arg Ala Glu Ala 210 215 220 Glu Arg Leu Arg Gln Arg Val Ala Glu LeuGlu Ala Gln Ala Ala Ser 225 230 235 240 Glu Val Thr Pro Thr Gly Glu LysGlu Gly Leu Gly Gln Gly Gln Ser 245 250 255 Leu Glu Gln Leu Glu Ala LeuVal Gln Thr Lys Asp Gln Glu Ile Gln 260 265 270 Thr Leu Lys Ser Gln ThrGly Gly Pro Arg Glu Ala Leu Glu Ala Ala 275 280 285 Glu Arg Glu Glu ThrGln Gln Lys Val Gln Thr Arg Asn Ala Glu Leu 290 295 300 Glu His Gln LeuArg Ala Met Glu Arg Ser Leu Glu Glu Ala Arg Ala 305 310 315 320 Glu ArgGlu Arg Ala Arg Ala Glu Val Gly Arg Ala Ala Gln Leu Leu 325 330 335 AspVal Ser Leu Phe Glu Leu Ser Glu Leu Arg Glu Gly Leu Ala Arg 340 345 350Leu Ala Glu Ala Ala Pro 355 <210> SEQ ID NO 13 <211> LENGTH: 6 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: core region for peptide binding <221> NAME/KEY: VARIANT<222> LOCATION: (1)..(6) <223> OTHER INFORMATION: Xaa is any amino acid<400> SEQUENCE: 13 Pro Pro Xaa Xaa Phe Arg 1 5 <210> SEQ ID NO 14 <211>LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: extended region for peptide binding<400> SEQUENCE: 14 Ala Leu Thr Pro Pro Ser Pro Phe Arg Asp 1 5 10 <210>SEQ ID NO 15 <211> LENGTH: 1415 <212> TYPE: DNA <213> ORGANISM: Rattusnorvegicus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1110) <400> SEQUENCE: 15 cac gcg tcc gtg gcg gag ctg cag cag ctgcag cag ttg cag gag ttc 48 His Ala Ser Val Ala Glu Leu Gln Gln Leu GlnGln Leu Gln Glu Phe 1 5 10 15 gat atc ccc acg ggc cgg gag gct ctg cggggc aac cac agc gcc ctg 96 Asp Ile Pro Thr Gly Arg Glu Ala Leu Arg GlyAsn His Ser Ala Leu 20 25 30 cta cgg gtg gcc aac tac tgt gag gat aac tacttg cag gcc aca gac 144 Leu Arg Val Ala Asn Tyr Cys Glu Asp Asn Tyr LeuGln Ala Thr Asp 35 40 45 aag cgg aag gcg ctg gaa gag acg atg gct ttc accacc cag gcc ctg 192 Lys Arg Lys Ala Leu Glu Glu Thr Met Ala Phe Thr ThrGln Ala Leu 50 55 60 gcc agt gta gcc tat caa gtg ggt aac ctg gcg ggg cacacg ctt cga 240 Ala Ser Val Ala Tyr Gln Val Gly Asn Leu Ala Gly His ThrLeu Arg 65 70 75 80 atg ctg gat cta cag ggt gct gcc ctg cgg cag gtg gaagcc aag atg 288 Met Leu Asp Leu Gln Gly Ala Ala Leu Arg Gln Val Glu AlaLys Met 85 90 95 agc aca ctg ggc cag atg gtg aac atg cac ctg gag aaa gtagcc aga 336 Ser Thr Leu Gly Gln Met Val Asn Met His Leu Glu Lys Val AlaArg 100 105 110 agg gag att ggc acg ttg gcc act gtc gtg cgg ctg ccc cctagc cag 384 Arg Glu Ile Gly Thr Leu Ala Thr Val Val Arg Leu Pro Pro SerGln 115 120 125 aag gtc atc cct cct gag agc ctg cct ccc ctc act ccc tactgc aga 432 Lys Val Ile Pro Pro Glu Ser Leu Pro Pro Leu Thr Pro Tyr CysArg 130 135 140 aaa ccc ctc aac ttt gcc tgc ttg gat gat gtt ggc cat ggagtc aag 480 Lys Pro Leu Asn Phe Ala Cys Leu Asp Asp Val Gly His Gly ValLys 145 150 155 160 gac ttg agc aca cag ctg tca cgg acc ggg acc ctg tctcgc aag agc 528 Asp Leu Ser Thr Gln Leu Ser Arg Thr Gly Thr Leu Ser ArgLys Ser 165 170 175 ata aag gcg ccc gct aca cct gcc tct gcc acg ctg gggaga cca ccc 576 Ile Lys Ala Pro Ala Thr Pro Ala Ser Ala Thr Leu Gly ArgPro Pro 180 185 190 cgg atc cct gag ccg gtg cag ctc cca gcg gtg cca gacggc aag ctc 624 Arg Ile Pro Glu Pro Val Gln Leu Pro Ala Val Pro Asp GlyLys Leu 195 200 205 tcc gct gcc tcc tct gtg tct tcc ttg gcc tcc gca ggcagt gca gaa 672 Ser Ala Ala Ser Ser Val Ser Ser Leu Ala Ser Ala Gly SerAla Glu 210 215 220 ggt gcc agt ggg atc ccc cag tcc aag gga cag gta gcacct gca acc 720 Gly Ala Ser Gly Ile Pro Gln Ser Lys Gly Gln Val Ala ProAla Thr 225 230 235 240 ccg cct cct cca cct ata gcg cct gta act cca cctcct cca cca ttg 768 Pro Pro Pro Pro Pro Ile Ala Pro Val Thr Pro Pro ProPro Pro Leu 245 250 255 cct gct gag atc ttc ttg ctg ccc cct ccg atg gaggag tcc cag ccc 816 Pro Ala Glu Ile Phe Leu Leu Pro Pro Pro Met Glu GluSer Gln Pro 260 265 270 cct ccg gaa aca gag ttg ccc ctg cct cct cct ccggct cta cag ggg 864 Pro Pro Glu Thr Glu Leu Pro Leu Pro Pro Pro Pro AlaLeu Gln Gly 275 280 285 gat gaa ctg ggg ctg ctg cct ccg cct cca cca ggtttt gga ccg gat 912 Asp Glu Leu Gly Leu Leu Pro Pro Pro Pro Pro Gly PheGly Pro Asp 290 295 300 gag ccc agc tgg gtc cct gct gcc tac ttg gag aaagtg gtg acg ctg 960 Glu Pro Ser Trp Val Pro Ala Ala Tyr Leu Glu Lys ValVal Thr Leu 305 310 315 320 tac cca tac acc cgg cag aag gac aat gag ctctcc ttt tct gaa gga 1008 Tyr Pro Tyr Thr Arg Gln Lys Asp Asn Glu Leu SerPhe Ser Glu Gly 325 330 335 acc gtc atc tgt gtc act cga cgc tac tca gatggc tgg tgt gag ggt 1056 Thr Val Ile Cys Val Thr Arg Arg Tyr Ser Asp GlyTrp Cys Glu Gly 340 345 350 gtc agc tca gag ggc act gga ttc ttc cca gggaac tat gtg gag ccc 1104 Val Ser Ser Glu Gly Thr Gly Phe Phe Pro Gly AsnTyr Val Glu Pro 355 360 365 agc tgc tgacagccca gatctgtccc tgcctctttggtgggcctct tgagccccaa 1160 Ser Cys 370 gaagccacct tccactcaaa gctggactaaggacctgtct acctcttggg ctgtgaactg 1220 tgttcagtcc cacacagcag taggaaggggtatgggatgg gctagagagt ggtggtactg 1280 aggacgattg ctccagatgg caagaacaaaacaaaacaaa ccaagaagtt aagtttaagc 1340 accttgccca gaggaccccc tagctcatgcaccgatcgcc agcattgaat aaaactgttg 1400 acctccagga ttgtt 1415 <210> SEQ IDNO 16 <211> LENGTH: 370 <212> TYPE: PRT <213> ORGANISM: Rattusnorvegicus <400> SEQUENCE: 16 His Ala Ser Val Ala Glu Leu Gln Gln LeuGln Gln Leu Gln Glu Phe 1 5 10 15 Asp Ile Pro Thr Gly Arg Glu Ala LeuArg Gly Asn His Ser Ala Leu 20 25 30 Leu Arg Val Ala Asn Tyr Cys Glu AspAsn Tyr Leu Gln Ala Thr Asp 35 40 45 Lys Arg Lys Ala Leu Glu Glu Thr MetAla Phe Thr Thr Gln Ala Leu 50 55 60 Ala Ser Val Ala Tyr Gln Val Gly AsnLeu Ala Gly His Thr Leu Arg 65 70 75 80 Met Leu Asp Leu Gln Gly Ala AlaLeu Arg Gln Val Glu Ala Lys Met 85 90 95 Ser Thr Leu Gly Gln Met Val AsnMet His Leu Glu Lys Val Ala Arg 100 105 110 Arg Glu Ile Gly Thr Leu AlaThr Val Val Arg Leu Pro Pro Ser Gln 115 120 125 Lys Val Ile Pro Pro GluSer Leu Pro Pro Leu Thr Pro Tyr Cys Arg 130 135 140 Lys Pro Leu Asn PheAla Cys Leu Asp Asp Val Gly His Gly Val Lys 145 150 155 160 Asp Leu SerThr Gln Leu Ser Arg Thr Gly Thr Leu Ser Arg Lys Ser 165 170 175 Ile LysAla Pro Ala Thr Pro Ala Ser Ala Thr Leu Gly Arg Pro Pro 180 185 190 ArgIle Pro Glu Pro Val Gln Leu Pro Ala Val Pro Asp Gly Lys Leu 195 200 205Ser Ala Ala Ser Ser Val Ser Ser Leu Ala Ser Ala Gly Ser Ala Glu 210 215220 Gly Ala Ser Gly Ile Pro Gln Ser Lys Gly Gln Val Ala Pro Ala Thr 225230 235 240 Pro Pro Pro Pro Pro Ile Ala Pro Val Thr Pro Pro Pro Pro ProLeu 245 250 255 Pro Ala Glu Ile Phe Leu Leu Pro Pro Pro Met Glu Glu SerGln Pro 260 265 270 Pro Pro Glu Thr Glu Leu Pro Leu Pro Pro Pro Pro AlaLeu Gln Gly 275 280 285 Asp Glu Leu Gly Leu Leu Pro Pro Pro Pro Pro GlyPhe Gly Pro Asp 290 295 300 Glu Pro Ser Trp Val Pro Ala Ala Tyr Leu GluLys Val Val Thr Leu 305 310 315 320 Tyr Pro Tyr Thr Arg Gln Lys Asp AsnGlu Leu Ser Phe Ser Glu Gly 325 330 335 Thr Val Ile Cys Val Thr Arg ArgTyr Ser Asp Gly Trp Cys Glu Gly 340 345 350 Val Ser Ser Glu Gly Thr GlyPhe Phe Pro Gly Asn Tyr Val Glu Pro 355 360 365 Ser Cys 370 <210> SEQ IDNO 17 <211> LENGTH: 3843 <212> TYPE: DNA <213> ORGANISM: Rattusnorvegicus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(3840) <400> SEQUENCE: 17 atg atg aca aac cga gat gga cgt gac tacttc atc aat cac atg aca 48 Met Met Thr Asn Arg Asp Gly Arg Asp Tyr PheIle Asn His Met Thr 1 5 10 15 cag gca atc cca ttt gat gac cct cgg tttgac agc tgc caa atc att 96 Gln Ala Ile Pro Phe Asp Asp Pro Arg Phe AspSer Cys Gln Ile Ile 20 25 30 ccc cca gct cca cgg aag gtg gag atg agg agggac cct gtg ctg ggc 144 Pro Pro Ala Pro Arg Lys Val Glu Met Arg Arg AspPro Val Leu Gly 35 40 45 ttt ggg ttc gtg gca ggg agt gaa aag cca gtg gtcgtt cga tcg gta 192 Phe Gly Phe Val Ala Gly Ser Glu Lys Pro Val Val ValArg Ser Val 50 55 60 aca cca ggt ggc cct tca gaa ggc aag ctg atc ccg ggagat caa att 240 Thr Pro Gly Gly Pro Ser Glu Gly Lys Leu Ile Pro Gly AspGln Ile 65 70 75 80 gta atg att aat gat gaa cca gtc agc gct gcg cca agagag agg gtc 288 Val Met Ile Asn Asp Glu Pro Val Ser Ala Ala Pro Arg GluArg Val 85 90 95 atc gac ctg gtc agg agc tgc aaa gaa tcg att ctg ttc actgtc atc 336 Ile Asp Leu Val Arg Ser Cys Lys Glu Ser Ile Leu Phe Thr ValIle 100 105 110 cag cct tat cct tct ccc aaa tca gca ttt att agt gct gctaaa aag 384 Gln Pro Tyr Pro Ser Pro Lys Ser Ala Phe Ile Ser Ala Ala LysLys 115 120 125 gca aga ttg aag tcc aat cca gtc aaa gta cgc ttt tcc gaagag gtc 432 Ala Arg Leu Lys Ser Asn Pro Val Lys Val Arg Phe Ser Glu GluVal 130 135 140 atc atc aat ggt cag gtg tcg gaa act gtt aaa gac aat tcactt ctt 480 Ile Ile Asn Gly Gln Val Ser Glu Thr Val Lys Asp Asn Ser LeuLeu 145 150 155 160 ttt atg cca aat gtt ttg aaa gtc tac ttg gaa aat ggacag acc aaa 528 Phe Met Pro Asn Val Leu Lys Val Tyr Leu Glu Asn Gly GlnThr Lys 165 170 175 tcc ttt cgc ttt gac tgc agc act tcc att aag gat gtcatc tta act 576 Ser Phe Arg Phe Asp Cys Ser Thr Ser Ile Lys Asp Val IleLeu Thr 180 185 190 ctg caa gag aag ctg tct atc aaa ggc att gag cac ttctct ctc atg 624 Leu Gln Glu Lys Leu Ser Ile Lys Gly Ile Glu His Phe SerLeu Met 195 200 205 ctg gag cag aga act gaa ggg gcc ggc acc aag ctg ctctta ctt cat 672 Leu Glu Gln Arg Thr Glu Gly Ala Gly Thr Lys Leu Leu LeuLeu His 210 215 220 gaa cag gag aca ctc act cag gtg aca cag agg ccg agttcc cat aag 720 Glu Gln Glu Thr Leu Thr Gln Val Thr Gln Arg Pro Ser SerHis Lys 225 230 235 240 atg agg tgt ctt ttc cga atc agt ttt gtt ccc aaggat ccc att gac 768 Met Arg Cys Leu Phe Arg Ile Ser Phe Val Pro Lys AspPro Ile Asp 245 250 255 ctg tta agg aga gat cca gtt gct ttc gag tat ctctat gtt cag agc 816 Leu Leu Arg Arg Asp Pro Val Ala Phe Glu Tyr Leu TyrVal Gln Ser 260 265 270 tgt aac gat gtc gtt cag gag cga ttt gga cca gagctg aaa tac gac 864 Cys Asn Asp Val Val Gln Glu Arg Phe Gly Pro Glu LeuLys Tyr Asp 275 280 285 att gcc ttg cgg ctg gcc gct tta caa atg tac attgct act gtc acc 912 Ile Ala Leu Arg Leu Ala Ala Leu Gln Met Tyr Ile AlaThr Val Thr 290 295 300 acc aaa cag acg cag aaa atc tcc ctc aag tac attgag aaa gaa tgg 960 Thr Lys Gln Thr Gln Lys Ile Ser Leu Lys Tyr Ile GluLys Glu Trp 305 310 315 320 gga cta gag act ttc ctt cca tct gct gta cttcag agc atg aaa gag 1008 Gly Leu Glu Thr Phe Leu Pro Ser Ala Val Leu GlnSer Met Lys Glu 325 330 335 aag aac atc aag aaa gcg ctc tcc cac ctt gtcaaa gca aat caa aac 1056 Lys Asn Ile Lys Lys Ala Leu Ser His Leu Val LysAla Asn Gln Asn 340 345 350 ttg gta cca ccg ggt aaa aag ctc tct gca ctacaa gct aag gtc cac 1104 Leu Val Pro Pro Gly Lys Lys Leu Ser Ala Leu GlnAla Lys Val His 355 360 365 tat ctc aag ttc ctc agt gac ctg cga cta tacggg ggc cgt gtg ttc 1152 Tyr Leu Lys Phe Leu Ser Asp Leu Arg Leu Tyr GlyGly Arg Val Phe 370 375 380 aag gca aca tta gtg cag gca gag aag cgc tcagaa gtg act ctt ctg 1200 Lys Ala Thr Leu Val Gln Ala Glu Lys Arg Ser GluVal Thr Leu Leu 385 390 395 400 gtg ggt ccc cgg tat ggc ata agc cat gtcata aac acc aaa acc aac 1248 Val Gly Pro Arg Tyr Gly Ile Ser His Val IleAsn Thr Lys Thr Asn 405 410 415 ctg gtg gct ctt tta gct gac ttc agc catgtc aac agg att gaa atg 1296 Leu Val Ala Leu Leu Ala Asp Phe Ser His ValAsn Arg Ile Glu Met 420 425 430 ttt act gaa gag gag agt ttg gtg agg gtggag ttg cat gtg ctc gat 1344 Phe Thr Glu Glu Glu Ser Leu Val Arg Val GluLeu His Val Leu Asp 435 440 445 gtg aag ccc att aca ctc ctt atg gag tcatca gat gcc atg aac ctg 1392 Val Lys Pro Ile Thr Leu Leu Met Glu Ser SerAsp Ala Met Asn Leu 450 455 460 gcc tgt ctg aca gct gga tac tac cgg ttgctc gtg gac tcc agg agg 1440 Ala Cys Leu Thr Ala Gly Tyr Tyr Arg Leu LeuVal Asp Ser Arg Arg 465 470 475 480 tca ata ttt aac atg gcc aac aag aaaaat gca ggc aca cag gac aca 1488 Ser Ile Phe Asn Met Ala Asn Lys Lys AsnAla Gly Thr Gln Asp Thr 485 490 495 gga acg gaa aat aaa ggc aag cat aatctc ctt ggt cct gac tgg aac 1536 Gly Thr Glu Asn Lys Gly Lys His Asn LeuLeu Gly Pro Asp Trp Asn 500 505 510 tgt atg ccc cag atg acg acc ttc attggc gaa ggg gaa caa gaa gcc 1584 Cys Met Pro Gln Met Thr Thr Phe Ile GlyGlu Gly Glu Gln Glu Ala 515 520 525 caa atc act tat ata gat tct aag cagaag gca gtt gag atg aca gac 1632 Gln Ile Thr Tyr Ile Asp Ser Lys Gln LysAla Val Glu Met Thr Asp 530 535 540 agc acc ttg tgt ccc aaa gag cac cggcac tta tat atc gac aac aca 1680 Ser Thr Leu Cys Pro Lys Glu His Arg HisLeu Tyr Ile Asp Asn Thr 545 550 555 560 tac agt tca gat gaa ctt agc cagccg ctg act cag cca ggt gat gca 1728 Tyr Ser Ser Asp Glu Leu Ser Gln ProLeu Thr Gln Pro Gly Asp Ala 565 570 575 ccc tgt gag gcc gac tat aga agccta gct cag cgg tcc ctt ttg acc 1776 Pro Cys Glu Ala Asp Tyr Arg Ser LeuAla Gln Arg Ser Leu Leu Thr 580 585 590 ctc tca gga cca gac act ctg aagaaa gca cag gaa tct ccg cga gga 1824 Leu Ser Gly Pro Asp Thr Leu Lys LysAla Gln Glu Ser Pro Arg Gly 595 600 605 gct aaa gtg tcc ttt att ttt ggagat ctt gcc tta gat gat ggc atg 1872 Ala Lys Val Ser Phe Ile Phe Gly AspLeu Ala Leu Asp Asp Gly Met 610 615 620 agt ccc cca act cta ggc tat gaaaga atg tta gat gag aat cca gaa 1920 Ser Pro Pro Thr Leu Gly Tyr Glu ArgMet Leu Asp Glu Asn Pro Glu 625 630 635 640 atg ctg gag aag cag agg aatctc tac atc agc agt gcc aat gat atg 1968 Met Leu Glu Lys Gln Arg Asn LeuTyr Ile Ser Ser Ala Asn Asp Met 645 650 655 aaa aac ctg gac ctc act ccagac aca gac agc atc cag ttt gtg gca 2016 Lys Asn Leu Asp Leu Thr Pro AspThr Asp Ser Ile Gln Phe Val Ala 660 665 670 aat tca gta tat gca aac ataggt gat gtg aag aac ttt gaa gcc cct 2064 Asn Ser Val Tyr Ala Asn Ile GlyAsp Val Lys Asn Phe Glu Ala Pro 675 680 685 gag gga ata gag gag ccc ctctta cat gac atc tgt tat gct gaa aac 2112 Glu Gly Ile Glu Glu Pro Leu LeuHis Asp Ile Cys Tyr Ala Glu Asn 690 695 700 aca gat gat gca gaa gat gaagat gag gtg agc tgc gag gag gat ctc 2160 Thr Asp Asp Ala Glu Asp Glu AspGlu Val Ser Cys Glu Glu Asp Leu 705 710 715 720 gtg gtg agt gaa atc aaccaa cca gcc atc ctt gac ctg tct ggg tca 2208 Val Val Ser Glu Ile Asn GlnPro Ala Ile Leu Asp Leu Ser Gly Ser 725 730 735 agt gat gat att att gacctt aca aca ctg cct cct cca gaa gga gat 2256 Ser Asp Asp Ile Ile Asp LeuThr Thr Leu Pro Pro Pro Glu Gly Asp 740 745 750 gac aat gag gat gac ttcctc ctg cgt tct ctg aac atg gcc att gct 2304 Asp Asn Glu Asp Asp Phe LeuLeu Arg Ser Leu Asn Met Ala Ile Ala 755 760 765 gct ccc cca cct ggt tttaga gac agt tct gat gaa gag gac act cag 2352 Ala Pro Pro Pro Gly Phe ArgAsp Ser Ser Asp Glu Glu Asp Thr Gln 770 775 780 agc cag gca aca tcc ttccat gag aac aaa gaa caa ggc agc agc ctg 2400 Ser Gln Ala Thr Ser Phe HisGlu Asn Lys Glu Gln Gly Ser Ser Leu 785 790 795 800 cag aat gag gag atccct gtg tcc ctc att gat gct gtg ccc acc agt 2448 Gln Asn Glu Glu Ile ProVal Ser Leu Ile Asp Ala Val Pro Thr Ser 805 810 815 gca gag ggc aag tgtgag aag gga ctg gac cct acc gtc gtt tcc aca 2496 Ala Glu Gly Lys Cys GluLys Gly Leu Asp Pro Thr Val Val Ser Thr 820 825 830 cta gaa gcc cta gaagct ctt tca gaa gaa cag cag aag agt gaa aat 2544 Leu Glu Ala Leu Glu AlaLeu Ser Glu Glu Gln Gln Lys Ser Glu Asn 835 840 845 tca ggt gta gcc atcttg cgg gct tat agt ccc gag tct tcc tca gac 2592 Ser Gly Val Ala Ile LeuArg Ala Tyr Ser Pro Glu Ser Ser Ser Asp 850 855 860 tcg ggc aat gag actaac tct tct gaa atg aca gag ggt tct gaa cta 2640 Ser Gly Asn Glu Thr AsnSer Ser Glu Met Thr Glu Gly Ser Glu Leu 865 870 875 880 gct gca gca cagaag cag tcg gaa agc ctc tcc cgc atg ttc ttg gcc 2688 Ala Ala Ala Gln LysGln Ser Glu Ser Leu Ser Arg Met Phe Leu Ala 885 890 895 act cat gaa ggttat cac cct ctg gca gaa gaa cag aca gag ttc ccc 2736 Thr His Glu Gly TyrHis Pro Leu Ala Glu Glu Gln Thr Glu Phe Pro 900 905 910 acc tcc aaa accccc tct gtg ggc ttg cct cca aag tcc tct cat ggc 2784 Thr Ser Lys Thr ProSer Val Gly Leu Pro Pro Lys Ser Ser His Gly 915 920 925 ctg gct gct cgccca gcg acc gac ctc cca ccc aaa gtt gtg cct tcc 2832 Leu Ala Ala Arg ProAla Thr Asp Leu Pro Pro Lys Val Val Pro Ser 930 935 940 aag cag atc cttcac tca gat cac atg gaa atg gag cca gaa acc atg 2880 Lys Gln Ile Leu HisSer Asp His Met Glu Met Glu Pro Glu Thr Met 945 950 955 960 gag acc aagtca gtc act gac tat ttt agc aaa ctg cac atg ggg tca 2928 Glu Thr Lys SerVal Thr Asp Tyr Phe Ser Lys Leu His Met Gly Ser 965 970 975 gtg gca tattcc tgt acc agc aaa agg aaa agc aag ctt gct gag gga 2976 Val Ala Tyr SerCys Thr Ser Lys Arg Lys Ser Lys Leu Ala Glu Gly 980 985 990 gag ggg aaatgc ccc ctg agt ggg aat gta cca ggg aaa aaa cag caa 3024 Glu Gly Lys CysPro Leu Ser Gly Asn Val Pro Gly Lys Lys Gln Gln 995 1000 1005 gga accaaa ata gca gag acg gag gag gac acc aaa ggc aaa gtt 3069 Gly Thr Lys IleAla Glu Thr Glu Glu Asp Thr Lys Gly Lys Val 1010 1015 1020 ggc act gtatct tca aga gac aat cca cac ctc agc act ttt aac 3114 Gly Thr Val Ser SerArg Asp Asn Pro His Leu Ser Thr Phe Asn 1025 1030 1035 ctg gag aga actgcc ttt cgc aag gac agc caa aga tgg tat gtg 3159 Leu Glu Arg Thr Ala PheArg Lys Asp Ser Gln Arg Trp Tyr Val 1040 1045 1050 gcc tct gat ggt ggggtg gta gag aaa agt gga gtg gaa gca cca 3204 Ala Ser Asp Gly Gly Val ValGlu Lys Ser Gly Val Glu Ala Pro 1055 1060 1065 gcc atg aaa gcc ttt cccaga ggt cct ggt ctg ggg aac aga gag 3249 Ala Met Lys Ala Phe Pro Arg GlyPro Gly Leu Gly Asn Arg Glu 1070 1075 1080 gct gaa ggg aaa gag gat ggcact atg gaa gga gag gct gat gat 3294 Ala Glu Gly Lys Glu Asp Gly Thr MetGlu Gly Glu Ala Asp Asp 1085 1090 1095 gct tca gga ctt ggt caa ggg gaacgc ttc ctg tca gat atg gcc 3339 Ala Ser Gly Leu Gly Gln Gly Glu Arg PheLeu Ser Asp Met Ala 1100 1105 1110 tgt gta gcc tca gcc aaa gac tta gacaac cct gaa gac act gac 3384 Cys Val Ala Ser Ala Lys Asp Leu Asp Asn ProGlu Asp Thr Asp 1115 1120 1125 tct ccc act tgt gac cat gcc act aag cttcct gag gct gaa gac 3429 Ser Pro Thr Cys Asp His Ala Thr Lys Leu Pro GluAla Glu Asp 1130 1135 1140 aat gtg gcc cgc ctt tgt gac tac cat ttg gccaag cga atg tca 3474 Asn Val Ala Arg Leu Cys Asp Tyr His Leu Ala Lys ArgMet Ser 1145 1150 1155 tcc ctg cag agt gag ggc cat ttt tct cta cag agctct caa ggc 3519 Ser Leu Gln Ser Glu Gly His Phe Ser Leu Gln Ser Ser GlnGly 1160 1165 1170 tct tca gtg gac aca ggc tgt ggc cca ggc agc agt agcagt gcc 3564 Ser Ser Val Asp Thr Gly Cys Gly Pro Gly Ser Ser Ser Ser Ala1175 1180 1185 tgt gcc act cct gtg gaa tcg ccc ctc tgc cca tcc atg ggaaag 3609 Cys Ala Thr Pro Val Glu Ser Pro Leu Cys Pro Ser Met Gly Lys1190 1195 1200 cac ctg att cca gat gct tct ggg aaa ggt ggg agt tac atttca 3654 His Leu Ile Pro Asp Ala Ser Gly Lys Gly Gly Ser Tyr Ile Ser1205 1210 1215 cca gag gag aga gtc gct ggt cat ccc aac cat gga gcc accttc 3699 Pro Glu Glu Arg Val Ala Gly His Pro Asn His Gly Ala Thr Phe1220 1225 1230 aag gaa ctg cac cca cag aca gaa ggg atg tgt cca cgc atgaca 3744 Lys Glu Leu His Pro Gln Thr Glu Gly Met Cys Pro Arg Met Thr1235 1240 1245 gtg cct gct ctg cac aca gcc att aat gcc gac ccc ctg tttggc 3789 Val Pro Ala Leu His Thr Ala Ile Asn Ala Asp Pro Leu Phe Gly1250 1255 1260 act ttg aga gat gga tgc cat cga ctg ccc aag att aag gaaacc 3834 Thr Leu Arg Asp Gly Cys His Arg Leu Pro Lys Ile Lys Glu Thr1265 1270 1275 aca gtg tag 3843 Thr Val 1280 <210> SEQ ID NO 18 <211>LENGTH: 1280 <212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400>SEQUENCE: 18 Met Met Thr Asn Arg Asp Gly Arg Asp Tyr Phe Ile Asn His MetThr 1 5 10 15 Gln Ala Ile Pro Phe Asp Asp Pro Arg Phe Asp Ser Cys GlnIle Ile 20 25 30 Pro Pro Ala Pro Arg Lys Val Glu Met Arg Arg Asp Pro ValLeu Gly 35 40 45 Phe Gly Phe Val Ala Gly Ser Glu Lys Pro Val Val Val ArgSer Val 50 55 60 Thr Pro Gly Gly Pro Ser Glu Gly Lys Leu Ile Pro Gly AspGln Ile 65 70 75 80 Val Met Ile Asn Asp Glu Pro Val Ser Ala Ala Pro ArgGlu Arg Val 85 90 95 Ile Asp Leu Val Arg Ser Cys Lys Glu Ser Ile Leu PheThr Val Ile 100 105 110 Gln Pro Tyr Pro Ser Pro Lys Ser Ala Phe Ile SerAla Ala Lys Lys 115 120 125 Ala Arg Leu Lys Ser Asn Pro Val Lys Val ArgPhe Ser Glu Glu Val 130 135 140 Ile Ile Asn Gly Gln Val Ser Glu Thr ValLys Asp Asn Ser Leu Leu 145 150 155 160 Phe Met Pro Asn Val Leu Lys ValTyr Leu Glu Asn Gly Gln Thr Lys 165 170 175 Ser Phe Arg Phe Asp Cys SerThr Ser Ile Lys Asp Val Ile Leu Thr 180 185 190 Leu Gln Glu Lys Leu SerIle Lys Gly Ile Glu His Phe Ser Leu Met 195 200 205 Leu Glu Gln Arg ThrGlu Gly Ala Gly Thr Lys Leu Leu Leu Leu His 210 215 220 Glu Gln Glu ThrLeu Thr Gln Val Thr Gln Arg Pro Ser Ser His Lys 225 230 235 240 Met ArgCys Leu Phe Arg Ile Ser Phe Val Pro Lys Asp Pro Ile Asp 245 250 255 LeuLeu Arg Arg Asp Pro Val Ala Phe Glu Tyr Leu Tyr Val Gln Ser 260 265 270Cys Asn Asp Val Val Gln Glu Arg Phe Gly Pro Glu Leu Lys Tyr Asp 275 280285 Ile Ala Leu Arg Leu Ala Ala Leu Gln Met Tyr Ile Ala Thr Val Thr 290295 300 Thr Lys Gln Thr Gln Lys Ile Ser Leu Lys Tyr Ile Glu Lys Glu Trp305 310 315 320 Gly Leu Glu Thr Phe Leu Pro Ser Ala Val Leu Gln Ser MetLys Glu 325 330 335 Lys Asn Ile Lys Lys Ala Leu Ser His Leu Val Lys AlaAsn Gln Asn 340 345 350 Leu Val Pro Pro Gly Lys Lys Leu Ser Ala Leu GlnAla Lys Val His 355 360 365 Tyr Leu Lys Phe Leu Ser Asp Leu Arg Leu TyrGly Gly Arg Val Phe 370 375 380 Lys Ala Thr Leu Val Gln Ala Glu Lys ArgSer Glu Val Thr Leu Leu 385 390 395 400 Val Gly Pro Arg Tyr Gly Ile SerHis Val Ile Asn Thr Lys Thr Asn 405 410 415 Leu Val Ala Leu Leu Ala AspPhe Ser His Val Asn Arg Ile Glu Met 420 425 430 Phe Thr Glu Glu Glu SerLeu Val Arg Val Glu Leu His Val Leu Asp 435 440 445 Val Lys Pro Ile ThrLeu Leu Met Glu Ser Ser Asp Ala Met Asn Leu 450 455 460 Ala Cys Leu ThrAla Gly Tyr Tyr Arg Leu Leu Val Asp Ser Arg Arg 465 470 475 480 Ser IlePhe Asn Met Ala Asn Lys Lys Asn Ala Gly Thr Gln Asp Thr 485 490 495 GlyThr Glu Asn Lys Gly Lys His Asn Leu Leu Gly Pro Asp Trp Asn 500 505 510Cys Met Pro Gln Met Thr Thr Phe Ile Gly Glu Gly Glu Gln Glu Ala 515 520525 Gln Ile Thr Tyr Ile Asp Ser Lys Gln Lys Ala Val Glu Met Thr Asp 530535 540 Ser Thr Leu Cys Pro Lys Glu His Arg His Leu Tyr Ile Asp Asn Thr545 550 555 560 Tyr Ser Ser Asp Glu Leu Ser Gln Pro Leu Thr Gln Pro GlyAsp Ala 565 570 575 Pro Cys Glu Ala Asp Tyr Arg Ser Leu Ala Gln Arg SerLeu Leu Thr 580 585 590 Leu Ser Gly Pro Asp Thr Leu Lys Lys Ala Gln GluSer Pro Arg Gly 595 600 605 Ala Lys Val Ser Phe Ile Phe Gly Asp Leu AlaLeu Asp Asp Gly Met 610 615 620 Ser Pro Pro Thr Leu Gly Tyr Glu Arg MetLeu Asp Glu Asn Pro Glu 625 630 635 640 Met Leu Glu Lys Gln Arg Asn LeuTyr Ile Ser Ser Ala Asn Asp Met 645 650 655 Lys Asn Leu Asp Leu Thr ProAsp Thr Asp Ser Ile Gln Phe Val Ala 660 665 670 Asn Ser Val Tyr Ala AsnIle Gly Asp Val Lys Asn Phe Glu Ala Pro 675 680 685 Glu Gly Ile Glu GluPro Leu Leu His Asp Ile Cys Tyr Ala Glu Asn 690 695 700 Thr Asp Asp AlaGlu Asp Glu Asp Glu Val Ser Cys Glu Glu Asp Leu 705 710 715 720 Val ValSer Glu Ile Asn Gln Pro Ala Ile Leu Asp Leu Ser Gly Ser 725 730 735 SerAsp Asp Ile Ile Asp Leu Thr Thr Leu Pro Pro Pro Glu Gly Asp 740 745 750Asp Asn Glu Asp Asp Phe Leu Leu Arg Ser Leu Asn Met Ala Ile Ala 755 760765 Ala Pro Pro Pro Gly Phe Arg Asp Ser Ser Asp Glu Glu Asp Thr Gln 770775 780 Ser Gln Ala Thr Ser Phe His Glu Asn Lys Glu Gln Gly Ser Ser Leu785 790 795 800 Gln Asn Glu Glu Ile Pro Val Ser Leu Ile Asp Ala Val ProThr Ser 805 810 815 Ala Glu Gly Lys Cys Glu Lys Gly Leu Asp Pro Thr ValVal Ser Thr 820 825 830 Leu Glu Ala Leu Glu Ala Leu Ser Glu Glu Gln GlnLys Ser Glu Asn 835 840 845 Ser Gly Val Ala Ile Leu Arg Ala Tyr Ser ProGlu Ser Ser Ser Asp 850 855 860 Ser Gly Asn Glu Thr Asn Ser Ser Glu MetThr Glu Gly Ser Glu Leu 865 870 875 880 Ala Ala Ala Gln Lys Gln Ser GluSer Leu Ser Arg Met Phe Leu Ala 885 890 895 Thr His Glu Gly Tyr His ProLeu Ala Glu Glu Gln Thr Glu Phe Pro 900 905 910 Thr Ser Lys Thr Pro SerVal Gly Leu Pro Pro Lys Ser Ser His Gly 915 920 925 Leu Ala Ala Arg ProAla Thr Asp Leu Pro Pro Lys Val Val Pro Ser 930 935 940 Lys Gln Ile LeuHis Ser Asp His Met Glu Met Glu Pro Glu Thr Met 945 950 955 960 Glu ThrLys Ser Val Thr Asp Tyr Phe Ser Lys Leu His Met Gly Ser 965 970 975 ValAla Tyr Ser Cys Thr Ser Lys Arg Lys Ser Lys Leu Ala Glu Gly 980 985 990Glu Gly Lys Cys Pro Leu Ser Gly Asn Val Pro Gly Lys Lys Gln Gln 995 10001005 Gly Thr Lys Ile Ala Glu Thr Glu Glu Asp Thr Lys Gly Lys Val 10101015 1020 Gly Thr Val Ser Ser Arg Asp Asn Pro His Leu Ser Thr Phe Asn1025 1030 1035 Leu Glu Arg Thr Ala Phe Arg Lys Asp Ser Gln Arg Trp TyrVal 1040 1045 1050 Ala Ser Asp Gly Gly Val Val Glu Lys Ser Gly Val GluAla Pro 1055 1060 1065 Ala Met Lys Ala Phe Pro Arg Gly Pro Gly Leu GlyAsn Arg Glu 1070 1075 1080 Ala Glu Gly Lys Glu Asp Gly Thr Met Glu GlyGlu Ala Asp Asp 1085 1090 1095 Ala Ser Gly Leu Gly Gln Gly Glu Arg PheLeu Ser Asp Met Ala 1100 1105 1110 Cys Val Ala Ser Ala Lys Asp Leu AspAsn Pro Glu Asp Thr Asp 1115 1120 1125 Ser Pro Thr Cys Asp His Ala ThrLys Leu Pro Glu Ala Glu Asp 1130 1135 1140 Asn Val Ala Arg Leu Cys AspTyr His Leu Ala Lys Arg Met Ser 1145 1150 1155 Ser Leu Gln Ser Glu GlyHis Phe Ser Leu Gln Ser Ser Gln Gly 1160 1165 1170 Ser Ser Val Asp ThrGly Cys Gly Pro Gly Ser Ser Ser Ser Ala 1175 1180 1185 Cys Ala Thr ProVal Glu Ser Pro Leu Cys Pro Ser Met Gly Lys 1190 1195 1200 His Leu IlePro Asp Ala Ser Gly Lys Gly Gly Ser Tyr Ile Ser 1205 1210 1215 Pro GluGlu Arg Val Ala Gly His Pro Asn His Gly Ala Thr Phe 1220 1225 1230 LysGlu Leu His Pro Gln Thr Glu Gly Met Cys Pro Arg Met Thr 1235 1240 1245Val Pro Ala Leu His Thr Ala Ile Asn Ala Asp Pro Leu Phe Gly 1250 12551260 Thr Leu Arg Asp Gly Cys His Arg Leu Pro Lys Ile Lys Glu Thr 12651270 1275 Thr Val 1280 <210> SEQ ID NO 19 <211> LENGTH: 1583 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (160)..(1257) <400> SEQUENCE: 19 ggcacgagtg agcatgcctgccctttgcaa gcaggtttgg gtctcacgca gaggaaacca 60 aaagcaataa gagggagggaaggcagagca accaatcaag ggcagggtga gactcaaaac 120 gagcgggctc cctggggagccagacagagg ctgggggtg atg gcg gag cta cag 174 Met Ala Glu Leu Gln 1 5 cagctg cag gag ttt gag atc ccc act ggc cgg gag gct ctg agg ggc 222 Gln LeuGln Glu Phe Glu Ile Pro Thr Gly Arg Glu Ala Leu Arg Gly 10 15 20 aac cacagt gcc ctg ctg cgg gtc gct gac tac tgc gag gac aac tat 270 Asn His SerAla Leu Leu Arg Val Ala Asp Tyr Cys Glu Asp Asn Tyr 25 30 35 gtg cag gccaca gac aag cgg aag gcg ctg gag gag acc atg gcc ttc 318 Val Gln Ala ThrAsp Lys Arg Lys Ala Leu Glu Glu Thr Met Ala Phe 40 45 50 act acc cag gcactg gcc agc gtg gcc tac cag gtg ggc aac ctg gcc 366 Thr Thr Gln Ala LeuAla Ser Val Ala Tyr Gln Val Gly Asn Leu Ala 55 60 65 ggg cac act ctg cgcatg ttg gac ctg cag ggg gcc gcc ctg cgg cag 414 Gly His Thr Leu Arg MetLeu Asp Leu Gln Gly Ala Ala Leu Arg Gln 70 75 80 85 gtg gaa gcc cgt gtaagc acg ctg ggc cag atg gtg aac atg cat atg 462 Val Glu Ala Arg Val SerThr Leu Gly Gln Met Val Asn Met His Met 90 95 100 gag aag gtg gcc cgaagg gag atc ggc acc tta gcc act gtc caa cgg 510 Glu Lys Val Ala Arg ArgGlu Ile Gly Thr Leu Ala Thr Val Gln Arg 105 110 115 ctg ccc ccc ggc cagaag gtc atc gcc cca gag aac cta ccc cct ctc 558 Leu Pro Pro Gly Gln LysVal Ile Ala Pro Glu Asn Leu Pro Pro Leu 120 125 130 acg ccc tac tgc aggaga acc ctc aac ttt ggc tgc ctg gac gac att 606 Thr Pro Tyr Cys Arg ArgThr Leu Asn Phe Gly Cys Leu Asp Asp Ile 135 140 145 ggc cat ggg atc aaggac ctc agc acg cag ctg tca aga aca ggc acc 654 Gly His Gly Ile Lys AspLeu Ser Thr Gln Leu Ser Arg Thr Gly Thr 150 155 160 165 ctg tct cga aagagc atc aag gcc cct gcc aca ccc gcc tcc gcc acc 702 Leu Ser Arg Lys SerIle Lys Ala Pro Ala Thr Pro Ala Ser Ala Thr 170 175 180 ttg ggg aga ccaccc cgg att ccc gag cca gtg cac ctg ccg gtg gtg 750 Leu Gly Arg Pro ProArg Ile Pro Glu Pro Val His Leu Pro Val Val 185 190 195 ccc gac ggc agactc tcc gcc gcc tcc tct gcg tct tcc ctg gcc tcg 798 Pro Asp Gly Arg LeuSer Ala Ala Ser Ser Ala Ser Ser Leu Ala Ser 200 205 210 gcc ggc agc gccgaa ggt gtc ggt ggg gcc ccc acg ccc aag ggg cag 846 Ala Gly Ser Ala GluGly Val Gly Gly Ala Pro Thr Pro Lys Gly Gln 215 220 225 gca gca cct ccagcc cca cct ctc ccc agc tcc ttg gac cca cct cct 894 Ala Ala Pro Pro AlaPro Pro Leu Pro Ser Ser Leu Asp Pro Pro Pro 230 235 240 245 cca cca gcagcc gtc gag gtg ttc cag cgg cct ccc acg ctg gag gag 942 Pro Pro Ala AlaVal Glu Val Phe Gln Arg Pro Pro Thr Leu Glu Glu 250 255 260 ttg tcc ccaccc cca ccg gac gaa gag ctg ccc ctg cca ctg gac ctg 990 Leu Ser Pro ProPro Pro Asp Glu Glu Leu Pro Leu Pro Leu Asp Leu 265 270 275 cct cct cctcca ccc ctg gat gga gat gaa ttg ggg ctg cct cca ccc 1038 Pro Pro Pro ProPro Leu Asp Gly Asp Glu Leu Gly Leu Pro Pro Pro 280 285 290 cca cca ggattt ggg cct gat gag ccc agc tgg gtg cct gcc tca tac 1086 Pro Pro Gly PheGly Pro Asp Glu Pro Ser Trp Val Pro Ala Ser Tyr 295 300 305 ttg gag aaagtg gtg aca ctg tac cca tac acc agc cag aag gac aat 1134 Leu Glu Lys ValVal Thr Leu Tyr Pro Tyr Thr Ser Gln Lys Asp Asn 310 315 320 325 gag ctctcc ttc tct gag ggc act gtc atc tgt gtc act cgc cgc tac 1182 Glu Leu SerPhe Ser Glu Gly Thr Val Ile Cys Val Thr Arg Arg Tyr 330 335 340 tcc gatggc tgg tgc gag ggc gtc agc tca gag ggg act gga ttc ttc 1230 Ser Asp GlyTrp Cys Glu Gly Val Ser Ser Glu Gly Thr Gly Phe Phe 345 350 355 cct gggaac tat gtg gag ccc agc tgc tgacagccca gggctctctg 1277 Pro Gly Asn TyrVal Glu Pro Ser Cys 360 365 ggcagctgat gtctgcactg agtgggtttc atgagccccaagccaaaacc agctccagtc 1337 acagctggac tgggtctgcc cacctcttgg gctgtgagctgtgttctgtc cttcctccca 1397 tcggagggag aaggggtcct ggggagagag aatttatccagaggcctgct gcagatgggg 1457 aagagctgga aaccaagaag tttgtcaaca gaggacccctactccatgca ggacagggtc 1517 tcctgctgca agtcccaact ttgaataaaa cagatgatgtcctgtgaaaa aaaaaaaaaa 1577 aaaaaa 1583 <210> SEQ ID NO 20 <211> LENGTH:366 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 MetAla Glu Leu Gln Gln Leu Gln Glu Phe Glu Ile Pro Thr Gly Arg 1 5 10 15Glu Ala Leu Arg Gly Asn His Ser Ala Leu Leu Arg Val Ala Asp Tyr 20 25 30Cys Glu Asp Asn Tyr Val Gln Ala Thr Asp Lys Arg Lys Ala Leu Glu 35 40 45Glu Thr Met Ala Phe Thr Thr Gln Ala Leu Ala Ser Val Ala Tyr Gln 50 55 60Val Gly Asn Leu Ala Gly His Thr Leu Arg Met Leu Asp Leu Gln Gly 65 70 7580 Ala Ala Leu Arg Gln Val Glu Ala Arg Val Ser Thr Leu Gly Gln Met 85 9095 Val Asn Met His Met Glu Lys Val Ala Arg Arg Glu Ile Gly Thr Leu 100105 110 Ala Thr Val Gln Arg Leu Pro Pro Gly Gln Lys Val Ile Ala Pro Glu115 120 125 Asn Leu Pro Pro Leu Thr Pro Tyr Cys Arg Arg Thr Leu Asn PheGly 130 135 140 Cys Leu Asp Asp Ile Gly His Gly Ile Lys Asp Leu Ser ThrGln Leu 145 150 155 160 Ser Arg Thr Gly Thr Leu Ser Arg Lys Ser Ile LysAla Pro Ala Thr 165 170 175 Pro Ala Ser Ala Thr Leu Gly Arg Pro Pro ArgIle Pro Glu Pro Val 180 185 190 His Leu Pro Val Val Pro Asp Gly Arg LeuSer Ala Ala Ser Ser Ala 195 200 205 Ser Ser Leu Ala Ser Ala Gly Ser AlaGlu Gly Val Gly Gly Ala Pro 210 215 220 Thr Pro Lys Gly Gln Ala Ala ProPro Ala Pro Pro Leu Pro Ser Ser 225 230 235 240 Leu Asp Pro Pro Pro ProPro Ala Ala Val Glu Val Phe Gln Arg Pro 245 250 255 Pro Thr Leu Glu GluLeu Ser Pro Pro Pro Pro Asp Glu Glu Leu Pro 260 265 270 Leu Pro Leu AspLeu Pro Pro Pro Pro Pro Leu Asp Gly Asp Glu Leu 275 280 285 Gly Leu ProPro Pro Pro Pro Gly Phe Gly Pro Asp Glu Pro Ser Trp 290 295 300 Val ProAla Ser Tyr Leu Glu Lys Val Val Thr Leu Tyr Pro Tyr Thr 305 310 315 320Ser Gln Lys Asp Asn Glu Leu Ser Phe Ser Glu Gly Thr Val Ile Cys 325 330335 Val Thr Arg Arg Tyr Ser Asp Gly Trp Cys Glu Gly Val Ser Ser Glu 340345 350 Gly Thr Gly Phe Phe Pro Gly Asn Tyr Val Glu Pro Ser Cys 355 360365 <210> SEQ ID NO 21 <211> LENGTH: 6935 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (498)..(3779) <400> SEQUENCE: 21 tttccagcca acgccaaacagtgactgttg acaatttcat attgtcatca ggggaaccaa 60 ggcttattca gatgcctatttcagaaccta ggacagttcc attgaaaagg cgcaggcgtt 120 cgggctggct gactagatggatcaggcctg gctgcctgat ggctatattc ctccttcctc 180 cctctccact tccatctcaacccttgaggc tgcatattga atagttggag aattcagtga 240 actaagagat gcaaatgcacagtacaaaat tcaaatgtcc aattcggggc agggctgcat 300 ctaactttaa tggcaaccactgcatgtgat gtctggggac tctatagata catggcctca 360 gaccctgaag acatctggattctgtcactg gattgttcac aaagtgaggc tgaactttcc 420 acaggacgaa gtcttcaggctggccgcctc cctcgggaac ctggggcttg agccaggtgc 480 cgccctatgg atgggag atgacg gca aac cga gat ggg cga gac tac ttc 530 Met Thr Ala Asn Arg Asp GlyArg Asp Tyr Phe 1 5 10 atc aat cac atg aca cag gca atc cct ttt gac gaccct cgg tta gag 578 Ile Asn His Met Thr Gln Ala Ile Pro Phe Asp Asp ProArg Leu Glu 15 20 25 agc tgc caa atc atc cct ccg gct cct cgg aag gtg gagatg aga agg 626 Ser Cys Gln Ile Ile Pro Pro Ala Pro Arg Lys Val Glu MetArg Arg 30 35 40 gac ccc gtg ctg gga ttt ggt ttt gtg gca ggc agt gaa aagcca gtg 674 Asp Pro Val Leu Gly Phe Gly Phe Val Ala Gly Ser Glu Lys ProVal 45 50 55 gtc gtt cgc tca gta aca cca ggt ggc ccc tct gaa ggc aag ctgatc 722 Val Val Arg Ser Val Thr Pro Gly Gly Pro Ser Glu Gly Lys Leu Ile60 65 70 75 ccg gga gat cag att gta atg att aat gat gaa ccg gtc agc gctgca 770 Pro Gly Asp Gln Ile Val Met Ile Asn Asp Glu Pro Val Ser Ala Ala80 85 90 ccc aga gag cgg gtc atc gat ctg gtc aga agc tgc aaa gaa tcg ata818 Pro Arg Glu Arg Val Ile Asp Leu Val Arg Ser Cys Lys Glu Ser Ile 95100 105 ctc ctc act gtc att cag cct tac cct tct ccc aaa tca gca ttt att866 Leu Leu Thr Val Ile Gln Pro Tyr Pro Ser Pro Lys Ser Ala Phe Ile 110115 120 agt gct gca aaa aag gca aga tta aag tcc aat cct gtc aaa gta cgc914 Ser Ala Ala Lys Lys Ala Arg Leu Lys Ser Asn Pro Val Lys Val Arg 125130 135 ttc tct gag gag gtc atc atc aac ggc caa gtg tcg gaa act gtt aag962 Phe Ser Glu Glu Val Ile Ile Asn Gly Gln Val Ser Glu Thr Val Lys 140145 150 155 gac aac tca ctt ctt ttt atg cca aat gtt ttg aaa gtc tat ctggaa 1010 Asp Asn Ser Leu Leu Phe Met Pro Asn Val Leu Lys Val Tyr Leu Glu160 165 170 aat ggg cag acc aaa tca ttt cgt ttt gac tgc agc act tcc attaag 1058 Asn Gly Gln Thr Lys Ser Phe Arg Phe Asp Cys Ser Thr Ser Ile Lys175 180 185 gat gtc atc tta acc ctt caa gag aag ctc tcc atc aaa ggc attgaa 1106 Asp Val Ile Leu Thr Leu Gln Glu Lys Leu Ser Ile Lys Gly Ile Glu190 195 200 cac ttc tct ctc atg ctg gag cag agg aca gaa ggg gct gga acgaag 1154 His Phe Ser Leu Met Leu Glu Gln Arg Thr Glu Gly Ala Gly Thr Lys205 210 215 ctg ctc ttg ctt cat gaa cag gag act cta act cag gtg aca cagagg 1202 Leu Leu Leu Leu His Glu Gln Glu Thr Leu Thr Gln Val Thr Gln Arg220 225 230 235 ccc agc tcc cat aag atg aga tgt ctt ttc cga att agc ttcgtc cca 1250 Pro Ser Ser His Lys Met Arg Cys Leu Phe Arg Ile Ser Phe ValPro 240 245 250 aaa gat cca att gac ctt tta agg aga gat cca gtt gct ttcgag tat 1298 Lys Asp Pro Ile Asp Leu Leu Arg Arg Asp Pro Val Ala Phe GluTyr 255 260 265 ctc tat gtt cag agt tgt aac gat gtg gtt cag gag cga tttggg ccg 1346 Leu Tyr Val Gln Ser Cys Asn Asp Val Val Gln Glu Arg Phe GlyPro 270 275 280 gag ctg aaa tat gac ata gcc ctg cgg ctg gcc gca tta caaatg tac 1394 Glu Leu Lys Tyr Asp Ile Ala Leu Arg Leu Ala Ala Leu Gln MetTyr 285 290 295 att gca acc gtt acc acc aag caa acg cag aaa atc tcc ctcaaa tac 1442 Ile Ala Thr Val Thr Thr Lys Gln Thr Gln Lys Ile Ser Leu LysTyr 300 305 310 315 atc gaa aaa gaa tgg gga tta gag act ttt ctt ccc tctgct gtg ctg 1490 Ile Glu Lys Glu Trp Gly Leu Glu Thr Phe Leu Pro Ser AlaVal Leu 320 325 330 caa agc atg aaa gag aag aac ata aag aaa gca ctt tcacac ctt gtc 1538 Gln Ser Met Lys Glu Lys Asn Ile Lys Lys Ala Leu Ser HisLeu Val 335 340 345 aaa gca aat caa aac ttg gta cca ccg ggt aaa aag ctctct gca cta 1586 Lys Ala Asn Gln Asn Leu Val Pro Pro Gly Lys Lys Leu SerAla Leu 350 355 360 caa gcc aag gtc cat tat ctc aag ttc ctc agt gac ctacga ttg tat 1634 Gln Ala Lys Val His Tyr Leu Lys Phe Leu Ser Asp Leu ArgLeu Tyr 365 370 375 ggg ggc cgt gtg ttc aag gca aca tta gtg cag gca gaaaag cgc tcg 1682 Gly Gly Arg Val Phe Lys Ala Thr Leu Val Gln Ala Glu LysArg Ser 380 385 390 395 gaa gtg act ctc ctg gtt ggg ccc cgg tat ggc ataagc cat gtc atc 1730 Glu Val Thr Leu Leu Val Gly Pro Arg Tyr Gly Ile SerHis Val Ile 400 405 410 aac acc aaa acc aat ctg gtg gct ctt tta gcc gacttt agc cac gtc 1778 Asn Thr Lys Thr Asn Leu Val Ala Leu Leu Ala Asp PheSer His Val 415 420 425 aac agg atc gaa atg ttt tcc gag gag gag agc ttggtg cgg gta gaa 1826 Asn Arg Ile Glu Met Phe Ser Glu Glu Glu Ser Leu ValArg Val Glu 430 435 440 ctc cac gtg cta gat gtg aag cct atc acg ctt ctgatg gaa tcc tca 1874 Leu His Val Leu Asp Val Lys Pro Ile Thr Leu Leu MetGlu Ser Ser 445 450 455 gat gcc atg aac ctg gcc tgc ttg acg gct gga tactac cgg ctg ctt 1922 Asp Ala Met Asn Leu Ala Cys Leu Thr Ala Gly Tyr TyrArg Leu Leu 460 465 470 475 gtt gat tcc agg agg tcg ata ttt aac atg gccaac aag aaa aac aca 1970 Val Asp Ser Arg Arg Ser Ile Phe Asn Met Ala AsnLys Lys Asn Thr 480 485 490 gcg acc cag gaa aca gga cct gaa aac aag gggaag cat aac ctc ctt 2018 Ala Thr Gln Glu Thr Gly Pro Glu Asn Lys Gly LysHis Asn Leu Leu 495 500 505 ggc cca gat tgg aac tgt ata ccc caa atg accacc ttt att ggc gaa 2066 Gly Pro Asp Trp Asn Cys Ile Pro Gln Met Thr ThrPhe Ile Gly Glu 510 515 520 ggg gaa caa gaa gcc cag ata aca tac ata gattca aag cag aag acg 2114 Gly Glu Gln Glu Ala Gln Ile Thr Tyr Ile Asp SerLys Gln Lys Thr 525 530 535 gtg gag atc aca gac agc acc atg tgt cca aaagag cac cgg cac ttg 2162 Val Glu Ile Thr Asp Ser Thr Met Cys Pro Lys GluHis Arg His Leu 540 545 550 555 tac ata gac aat gcc tat agt tca gat ggactt aac cag cag ctg agc 2210 Tyr Ile Asp Asn Ala Tyr Ser Ser Asp Gly LeuAsn Gln Gln Leu Ser 560 565 570 cag ccc ggg gag gcc ccc tgt gag gca gactac aga agt cta gct cag 2258 Gln Pro Gly Glu Ala Pro Cys Glu Ala Asp TyrArg Ser Leu Ala Gln 575 580 585 cgg tcc cta ttg acc ctc tca gga cca gaaact ctg aag aaa gca cag 2306 Arg Ser Leu Leu Thr Leu Ser Gly Pro Glu ThrLeu Lys Lys Ala Gln 590 595 600 gaa tct ccg aga gga gct aaa gtg tcc tttatt ttt gga gac ttc gcc 2354 Glu Ser Pro Arg Gly Ala Lys Val Ser Phe IlePhe Gly Asp Phe Ala 605 610 615 ttg gat gat ggt att agt ccc cca acc cttggc tat gaa acg cta cta 2402 Leu Asp Asp Gly Ile Ser Pro Pro Thr Leu GlyTyr Glu Thr Leu Leu 620 625 630 635 gat gag ggt cct gaa atg ctg gag aagcag aga aat ctc tac att ggc 2450 Asp Glu Gly Pro Glu Met Leu Glu Lys GlnArg Asn Leu Tyr Ile Gly 640 645 650 agt gcc aat gac atg aag ggc ctg gatctc act cca gag gca gag ggc 2498 Ser Ala Asn Asp Met Lys Gly Leu Asp LeuThr Pro Glu Ala Glu Gly 655 660 665 atc cag ttt gtg gaa aat tct gtt tatgca aac ata ggc gat gtg aag 2546 Ile Gln Phe Val Glu Asn Ser Val Tyr AlaAsn Ile Gly Asp Val Lys 670 675 680 agc ttc cag gcc gcg gag ggg atc gaggaa ccc ctc ttg cat gac atc 2594 Ser Phe Gln Ala Ala Glu Gly Ile Glu GluPro Leu Leu His Asp Ile 685 690 695 tgt tat gca gaa aac act gat gac gcggag gac gag gac gag gtg agc 2642 Cys Tyr Ala Glu Asn Thr Asp Asp Ala GluAsp Glu Asp Glu Val Ser 700 705 710 715 tgc gag gag gac ctc gtg gtg ggggag atg aac cag ccg gcc atc ctc 2690 Cys Glu Glu Asp Leu Val Val Gly GluMet Asn Gln Pro Ala Ile Leu 720 725 730 aac ctg tct ggg tca agc gat gacatc att gac ctc aca tcc ctg ccc 2738 Asn Leu Ser Gly Ser Ser Asp Asp IleIle Asp Leu Thr Ser Leu Pro 735 740 745 cct cca gaa ggt gat gac aat gaggat gac ttc ctg ttg cgt tcc ttg 2786 Pro Pro Glu Gly Asp Asp Asn Glu AspAsp Phe Leu Leu Arg Ser Leu 750 755 760 aac atg gcc att gcc gca ccc ccacct ggc ttt aga gac agt tca gat 2834 Asn Met Ala Ile Ala Ala Pro Pro ProGly Phe Arg Asp Ser Ser Asp 765 770 775 gaa gag gac tct cag agc cag gcagct tcc ttc ccc gag gac aag gag 2882 Glu Glu Asp Ser Gln Ser Gln Ala AlaSer Phe Pro Glu Asp Lys Glu 780 785 790 795 aaa ggc agc agc ctg caa aatgat gag atc ccc gtg tcc ctc att gac 2930 Lys Gly Ser Ser Leu Gln Asn AspGlu Ile Pro Val Ser Leu Ile Asp 800 805 810 gct gtg ccc acc agc gcc gaaggc aag tgt gag aag gga ctg gat aat 2978 Ala Val Pro Thr Ser Ala Glu GlyLys Cys Glu Lys Gly Leu Asp Asn 815 820 825 gcc gtc gtc tcc acg ctg ggagct cta gag gct cta tcc gtg tca gaa 3026 Ala Val Val Ser Thr Leu Gly AlaLeu Glu Ala Leu Ser Val Ser Glu 830 835 840 gaa cag cag acc agt gac aattca ggt gta gcc atc ttg cgg gct tat 3074 Glu Gln Gln Thr Ser Asp Asn SerGly Val Ala Ile Leu Arg Ala Tyr 845 850 855 agt cct gag tct tcg tca gactcg ggc aat gaa act aac tct tct gaa 3122 Ser Pro Glu Ser Ser Ser Asp SerGly Asn Glu Thr Asn Ser Ser Glu 860 865 870 875 atg act gag agt tct gaactg gcc aca gca caa aaa cag tca gaa aac 3170 Met Thr Glu Ser Ser Glu LeuAla Thr Ala Gln Lys Gln Ser Glu Asn 880 885 890 ctc tcc cgc atg ttc ttggcc act cac gaa ggc tac cac ccc ctt gca 3218 Leu Ser Arg Met Phe Leu AlaThr His Glu Gly Tyr His Pro Leu Ala 895 900 905 gaa gag cag acc gag ttcccg gcc tcc aag acc ccc gct ggg ggc ttg 3266 Glu Glu Gln Thr Glu Phe ProAla Ser Lys Thr Pro Ala Gly Gly Leu 910 915 920 cct cca aag tcc tcg cacgcc ctg gct gct agg cca gca acc gac ctc 3314 Pro Pro Lys Ser Ser His AlaLeu Ala Ala Arg Pro Ala Thr Asp Leu 925 930 935 ccg ccc aaa gtt gtg ccttcc aag cag tta ctt cac tca gac cac atg 3362 Pro Pro Lys Val Val Pro SerLys Gln Leu Leu His Ser Asp His Met 940 945 950 955 gag atg gag cct gaaact atg gag act aag tcg gtc act gac tat ttt 3410 Glu Met Glu Pro Glu ThrMet Glu Thr Lys Ser Val Thr Asp Tyr Phe 960 965 970 agc aaa ctg cac atgggg tcg gtg gca tac tcc tgc act agc aaa agg 3458 Ser Lys Leu His Met GlySer Val Ala Tyr Ser Cys Thr Ser Lys Arg 975 980 985 aaa agc aag ctg gccgat ggt gag ggg aag gca ccc cct aat ggg aac 3506 Lys Ser Lys Leu Ala AspGly Glu Gly Lys Ala Pro Pro Asn Gly Asn 990 995 1000 aca aca gga aaa aaacag cag ggg acc aaa acg gca gag atg gag 3551 Thr Thr Gly Lys Lys Gln GlnGly Thr Lys Thr Ala Glu Met Glu 1005 1010 1015 gag gag gcc agt ggt aaattt ggt act gtg tct tca cga gac agt 3596 Glu Glu Ala Ser Gly Lys Phe GlyThr Val Ser Ser Arg Asp Ser 1020 1025 1030 caa cac ctg agc act ttt aatctg gag aga act gcc ttt cgc aag 3641 Gln His Leu Ser Thr Phe Asn Leu GluArg Thr Ala Phe Arg Lys 1035 1040 1045 gac agt caa aga tgg tat gtg gccact gaa ggt ggg atg gct gaa 3686 Asp Ser Gln Arg Trp Tyr Val Ala Thr GluGly Gly Met Ala Glu 1050 1055 1060 aaa aag tgg att aga agc agc aac agggaa aac ctt tcc aag agc 3731 Lys Lys Trp Ile Arg Ser Ser Asn Arg Glu AsnLeu Ser Lys Ser 1065 1070 1075 ttc tgg tct tgg ggc aag gga ggc cga agggaa gga aga agg agc 3776 Phe Trp Ser Trp Gly Lys Gly Gly Arg Arg Glu GlyArg Arg Ser 1080 1085 1090 tcc tgatggagaa accagtgatg gctcaggacttggtcaaggg gaccgcttct 3829 Ser taactgacgt gacctgtgca tcttcagccaaagacttaga taacccagag gacgctgact 3889 cgtccacctg cgaccatcct tccaagcttcctgaggctga tgagagtgtg gcccgccttt 3949 gtgactacca cttggccaag cggatgtcatcactgcaaag cgagggccat ttttctctgc 4009 agagctccca aggctcttca gtggatgcaggctgtggcac aggcagcagt ggcagtgcct 4069 gtgccacacc cgtggagtcg ccgctctgcccctccctggg gaagcacttg attcctgacg 4129 cttctgggaa aggcgtgaat tacattccttcagaggagag agcccctggg cttcccaacc 4189 acggagccac ctttaaggaa ctgcacccacagacagaagg gatgtgtcca cggatgacag 4249 tgcctgctct gcacacagcc attaacaccgaacccctgtt tggcacattg agagatggat 4309 gccatcggct ccccaagatt aaggaaaccacagtgtagct ttgacagagc ctgggaagga 4369 gagacgagga ggcatgcctt cagcttggtctcaacatcct gaagctgatc ccatcctgct 4429 accatcaaac attcactcgg aatcaaaggtgccaattcca aatcaagacc ctaatgattt 4489 ctcccaagca aatcaggcat acggagaggctgtgagctgg cggccaccgg atctgagagg 4549 ggggagcctc aggacacctc ccagccagaaggctctgaga catagcagca gtatcctctc 4609 cggatctgtc gatttggaga ccttccgagagagaaccaag ggtgcagtca gcttaaagtg 4669 tccaggcatc acagaagcac aggaggccagttctgaaagg cgagcagaac tccccctggg 4729 gaggaagctc accaaaagtt tttcccaaagctcaatgcac ttgagctctg aggggaggtt 4789 tcacaaaagg tccccagtgg ctcataaagactcaaagctg tataggacat tacccttgcg 4849 gaagctggag ggcagcaatt ggagatgccggggacccttc agctattgct tcctgaaccg 4909 agggcaggat gaagatggtg aggaagaagaggagagggga gaggccaccg tccaggtctc 4969 ttgcctctat agaccacagg tgactcaagccatgccagaa ccaagcagcc catgcctggc 5029 tgtggcgatt cagaagcaac gaggggagctatccagaggg tcagtgctga aggtctgggc 5089 agaagacctg cgagacccag atgacttggacttcagcaac ctggcttttg atgcccggat 5149 tgcaagaata aatgccctaa aggagagcacatatgcaatg cctgatgggt tccttgcagc 5209 ccaaaatgat gccaatgagc tgctctgtctcgtcagggca accaaggaga agagggagga 5269 gtcacgccct gaagcgtacg accttacactttctcagtac aagcaactgt tatccattga 5329 gtccagacag ttgggaagtg cctgtaggaaaatggcgatg gctgagaaaa gcccggagga 5389 gatgctccta gctatgactt ccagctttcaagtgctctgt tgcctaacag aagcttgcat 5449 gcgattagtt aaagtcgtga actcagaaacacagcggcag gaaattgtag ggaagatcga 5509 tgaagtggtc ataaattaca tttgtctactgaaagctgcc gaagcagcca ctggaaagaa 5569 ccctggggac cctaatgttg gactctcggcgcgacactca accaccatgg ccgctctcgt 5629 aagcacactg acacgttctc tcaagaggcttttaaacaaa taaatatgga agtcacgtca 5689 taatctacct ttgcaaagcc atacatgaacttttatttac tttgtgtgta tgatgaacag 5749 atgtctcctt tcttctctct gtatattttgttattttata taaaatagga gataaaagtc 5809 acactgatga aatgttgaaa tgtactaatcagatgtattc tgtttatatt atacatatat 5869 atacacgtaa aagaaatatc caagaaagtgatgacatttg gctatttttc atatagttaa 5929 aactccaggt atatgatgtg aaattttaaattctaccatg ttagagcaaa acaatgaatc 5989 ctatcccctt tctttccaag tagctacttggaaaccatat cattcatatt tagaagtaaa 6049 acacaaaaca aaaaagagag agaaaagaaaagaaatcaca atgtatataa aacagtactt 6109 atgttttaaa attatgattt ttaagcattggaaatagcaa aaagacattt aaaattcaag 6169 aagctattat gaattactag agaatatatctgtaataaat taattttttg ctcatagtat 6229 ttggttactg gatgctttct tccaagaatcccacatattt aatttgggtt tttgctactg 6289 gggctacaaa ttggtgggga tggattctactgtgtcagca caaatgctct tcacagtggt 6349 tctagcattt aaaaaacttc ccggggagaagaacagaggg gatgatgggc agtttcctag 6409 gtaacaccta gagttataga atatctcattacataaaatg tatggaatta ataataccaa 6469 aattaattat ttgatggaaa gatctgctttgactaaatgt caaaaatctg caaaccaaag 6529 acattatctt cccctcatcc caactcaactacgaaactta aaattccctt tagagtgata 6589 ggacatttag taaagtattt gcaaacttaaaaaaaggaac atttaatgat catcaaaatt 6649 aagtacagat tcagtaatgt agaccagaccacacaccagc acctgtgagt ctcatctcag 6709 atcacagctc tcagcatagg gcttcatgcatcaccgcctc tacagaggct aaggctgcca 6769 gtcaaatttg gaattatagc gtagtactgggacaaaatct caaatcttgg atgttccaga 6829 aaatcaggga gtgatggcta ctgtaatcatgggagccatg agtaaatagt taagtattta 6889 ttaaataaat acttaatctg gattggctgataaaaatatg aaatct 6935 <210> SEQ ID NO 22 <211> LENGTH: 1094 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 Met Thr Ala Asn ArgAsp Gly Arg Asp Tyr Phe Ile Asn His Met Thr 1 5 10 15 Gln Ala Ile ProPhe Asp Asp Pro Arg Leu Glu Ser Cys Gln Ile Ile 20 25 30 Pro Pro Ala ProArg Lys Val Glu Met Arg Arg Asp Pro Val Leu Gly 35 40 45 Phe Gly Phe ValAla Gly Ser Glu Lys Pro Val Val Val Arg Ser Val 50 55 60 Thr Pro Gly GlyPro Ser Glu Gly Lys Leu Ile Pro Gly Asp Gln Ile 65 70 75 80 Val Met IleAsn Asp Glu Pro Val Ser Ala Ala Pro Arg Glu Arg Val 85 90 95 Ile Asp LeuVal Arg Ser Cys Lys Glu Ser Ile Leu Leu Thr Val Ile 100 105 110 Gln ProTyr Pro Ser Pro Lys Ser Ala Phe Ile Ser Ala Ala Lys Lys 115 120 125 AlaArg Leu Lys Ser Asn Pro Val Lys Val Arg Phe Ser Glu Glu Val 130 135 140Ile Ile Asn Gly Gln Val Ser Glu Thr Val Lys Asp Asn Ser Leu Leu 145 150155 160 Phe Met Pro Asn Val Leu Lys Val Tyr Leu Glu Asn Gly Gln Thr Lys165 170 175 Ser Phe Arg Phe Asp Cys Ser Thr Ser Ile Lys Asp Val Ile LeuThr 180 185 190 Leu Gln Glu Lys Leu Ser Ile Lys Gly Ile Glu His Phe SerLeu Met 195 200 205 Leu Glu Gln Arg Thr Glu Gly Ala Gly Thr Lys Leu LeuLeu Leu His 210 215 220 Glu Gln Glu Thr Leu Thr Gln Val Thr Gln Arg ProSer Ser His Lys 225 230 235 240 Met Arg Cys Leu Phe Arg Ile Ser Phe ValPro Lys Asp Pro Ile Asp 245 250 255 Leu Leu Arg Arg Asp Pro Val Ala PheGlu Tyr Leu Tyr Val Gln Ser 260 265 270 Cys Asn Asp Val Val Gln Glu ArgPhe Gly Pro Glu Leu Lys Tyr Asp 275 280 285 Ile Ala Leu Arg Leu Ala AlaLeu Gln Met Tyr Ile Ala Thr Val Thr 290 295 300 Thr Lys Gln Thr Gln LysIle Ser Leu Lys Tyr Ile Glu Lys Glu Trp 305 310 315 320 Gly Leu Glu ThrPhe Leu Pro Ser Ala Val Leu Gln Ser Met Lys Glu 325 330 335 Lys Asn IleLys Lys Ala Leu Ser His Leu Val Lys Ala Asn Gln Asn 340 345 350 Leu ValPro Pro Gly Lys Lys Leu Ser Ala Leu Gln Ala Lys Val His 355 360 365 TyrLeu Lys Phe Leu Ser Asp Leu Arg Leu Tyr Gly Gly Arg Val Phe 370 375 380Lys Ala Thr Leu Val Gln Ala Glu Lys Arg Ser Glu Val Thr Leu Leu 385 390395 400 Val Gly Pro Arg Tyr Gly Ile Ser His Val Ile Asn Thr Lys Thr Asn405 410 415 Leu Val Ala Leu Leu Ala Asp Phe Ser His Val Asn Arg Ile GluMet 420 425 430 Phe Ser Glu Glu Glu Ser Leu Val Arg Val Glu Leu His ValLeu Asp 435 440 445 Val Lys Pro Ile Thr Leu Leu Met Glu Ser Ser Asp AlaMet Asn Leu 450 455 460 Ala Cys Leu Thr Ala Gly Tyr Tyr Arg Leu Leu ValAsp Ser Arg Arg 465 470 475 480 Ser Ile Phe Asn Met Ala Asn Lys Lys AsnThr Ala Thr Gln Glu Thr 485 490 495 Gly Pro Glu Asn Lys Gly Lys His AsnLeu Leu Gly Pro Asp Trp Asn 500 505 510 Cys Ile Pro Gln Met Thr Thr PheIle Gly Glu Gly Glu Gln Glu Ala 515 520 525 Gln Ile Thr Tyr Ile Asp SerLys Gln Lys Thr Val Glu Ile Thr Asp 530 535 540 Ser Thr Met Cys Pro LysGlu His Arg His Leu Tyr Ile Asp Asn Ala 545 550 555 560 Tyr Ser Ser AspGly Leu Asn Gln Gln Leu Ser Gln Pro Gly Glu Ala 565 570 575 Pro Cys GluAla Asp Tyr Arg Ser Leu Ala Gln Arg Ser Leu Leu Thr 580 585 590 Leu SerGly Pro Glu Thr Leu Lys Lys Ala Gln Glu Ser Pro Arg Gly 595 600 605 AlaLys Val Ser Phe Ile Phe Gly Asp Phe Ala Leu Asp Asp Gly Ile 610 615 620Ser Pro Pro Thr Leu Gly Tyr Glu Thr Leu Leu Asp Glu Gly Pro Glu 625 630635 640 Met Leu Glu Lys Gln Arg Asn Leu Tyr Ile Gly Ser Ala Asn Asp Met645 650 655 Lys Gly Leu Asp Leu Thr Pro Glu Ala Glu Gly Ile Gln Phe ValGlu 660 665 670 Asn Ser Val Tyr Ala Asn Ile Gly Asp Val Lys Ser Phe GlnAla Ala 675 680 685 Glu Gly Ile Glu Glu Pro Leu Leu His Asp Ile Cys TyrAla Glu Asn 690 695 700 Thr Asp Asp Ala Glu Asp Glu Asp Glu Val Ser CysGlu Glu Asp Leu 705 710 715 720 Val Val Gly Glu Met Asn Gln Pro Ala IleLeu Asn Leu Ser Gly Ser 725 730 735 Ser Asp Asp Ile Ile Asp Leu Thr SerLeu Pro Pro Pro Glu Gly Asp 740 745 750 Asp Asn Glu Asp Asp Phe Leu LeuArg Ser Leu Asn Met Ala Ile Ala 755 760 765 Ala Pro Pro Pro Gly Phe ArgAsp Ser Ser Asp Glu Glu Asp Ser Gln 770 775 780 Ser Gln Ala Ala Ser PhePro Glu Asp Lys Glu Lys Gly Ser Ser Leu 785 790 795 800 Gln Asn Asp GluIle Pro Val Ser Leu Ile Asp Ala Val Pro Thr Ser 805 810 815 Ala Glu GlyLys Cys Glu Lys Gly Leu Asp Asn Ala Val Val Ser Thr 820 825 830 Leu GlyAla Leu Glu Ala Leu Ser Val Ser Glu Glu Gln Gln Thr Ser 835 840 845 AspAsn Ser Gly Val Ala Ile Leu Arg Ala Tyr Ser Pro Glu Ser Ser 850 855 860Ser Asp Ser Gly Asn Glu Thr Asn Ser Ser Glu Met Thr Glu Ser Ser 865 870875 880 Glu Leu Ala Thr Ala Gln Lys Gln Ser Glu Asn Leu Ser Arg Met Phe885 890 895 Leu Ala Thr His Glu Gly Tyr His Pro Leu Ala Glu Glu Gln ThrGlu 900 905 910 Phe Pro Ala Ser Lys Thr Pro Ala Gly Gly Leu Pro Pro LysSer Ser 915 920 925 His Ala Leu Ala Ala Arg Pro Ala Thr Asp Leu Pro ProLys Val Val 930 935 940 Pro Ser Lys Gln Leu Leu His Ser Asp His Met GluMet Glu Pro Glu 945 950 955 960 Thr Met Glu Thr Lys Ser Val Thr Asp TyrPhe Ser Lys Leu His Met 965 970 975 Gly Ser Val Ala Tyr Ser Cys Thr SerLys Arg Lys Ser Lys Leu Ala 980 985 990 Asp Gly Glu Gly Lys Ala Pro ProAsn Gly Asn Thr Thr Gly Lys Lys 995 1000 1005 Gln Gln Gly Thr Lys ThrAla Glu Met Glu Glu Glu Ala Ser Gly 1010 1015 1020 Lys Phe Gly Thr ValSer Ser Arg Asp Ser Gln His Leu Ser Thr 1025 1030 1035 Phe Asn Leu GluArg Thr Ala Phe Arg Lys Asp Ser Gln Arg Trp 1040 1045 1050 Tyr Val AlaThr Glu Gly Gly Met Ala Glu Lys Lys Trp Ile Arg 1055 1060 1065 Ser SerAsn Arg Glu Asn Leu Ser Lys Ser Phe Trp Ser Trp Gly 1070 1075 1080 LysGly Gly Arg Arg Glu Gly Arg Arg Ser Ser 1085 1090 <210> SEQ ID NO 23<211> LENGTH: 2139 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (806)..(1363) <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(2139) <223> OTHERINFORMATION: n is either a, c, g, or t <400> SEQUENCE: 23 agcggggctccattgtgctc ggcgggggcc gggaagccaa aggaggtggg ctcgggcccc 60 tgcgctgctccccggcggct gcgcccccag ctagctgcca gcctggaaat ggctccgctg 120 ctgctcctcgggaaaacgaa tcgatccttc ccagccttct ctgcctgctc tccacctcct 180 ctctgctccgagtcttagga ggacgaacat tcaaaggaca gattccaatg tggtgtgccg 240 tgcacatcgggagcggctgg ggtttgcact tcgagatttc ttctatataa tttttttttt 300 ttaaacgtaagggaggcagt agcattgctg cctgtaggat tttttattca agtgcacgtc 360 gcgttgggttgcacgntcca cccccaggga cctggtgtgg tgaaatttga acccaccgcc 420 ttagcccaaaaaggccgagt aacctggctg cctgagtgtc gtggaagacg tgagcgaaat 480 gaccagcgaactcatttttt atcagacttg ctgaagctgg cttttgcgtt ttttctacac 540 gtacgcttaattttgtggaa tagttaagtg ctatattctc cgcgcaacct tttcaaattc 600 caaatgtttgaacattttgg tgtcagcgcg agtgaaatca ttttaccgac aagaactaac 660 tgaattgtctgccttgttga gttgcctccg gaaaagatct cgggggtgga aaagcaactg 720 caaaataacagacggagaaa attccttgga agttatttct gtagcataag agcagaaact 780 tcagagcaagttttcattgg gcaaa atg ggg gag caa cct atc ttc agc act 832 Met Gly Glu GlnPro Ile Phe Ser Thr 1 5 cga gct cat gtc ttc cag att gac ccg aac aca aagaag aac tgg gta 880 Arg Ala His Val Phe Gln Ile Asp Pro Asn Thr Lys LysAsn Trp Val 10 15 20 25 ccc acc agc aag cat gca gtt act gta tct tat ttttat gac agc aca 928 Pro Thr Ser Lys His Ala Val Thr Val Ser Tyr Phe TyrAsp Ser Thr 30 35 40 aga aat gtg tat agg ata atc agt tta gat ggc tca aaggca ata ata 976 Arg Asn Val Tyr Arg Ile Ile Ser Leu Asp Gly Ser Lys AlaIle Ile 45 50 55 aat agc acc atc aca cca aac atg aca ttt act aaa aca tctcaa aag 1024 Asn Ser Thr Ile Thr Pro Asn Met Thr Phe Thr Lys Thr Ser GlnLys 60 65 70 ttt ggc caa tgg gct gat agc cgg gca aac act gtt tat gga ctggga 1072 Phe Gly Gln Trp Ala Asp Ser Arg Ala Asn Thr Val Tyr Gly Leu Gly75 80 85 ttc tcc tct gag cat cat ctt tca aaa ttc gca gaa aag ttt cag gaa1120 Phe Ser Ser Glu His His Leu Ser Lys Phe Ala Glu Lys Phe Gln Glu 9095 100 105 ttt aag gaa gct gct cgg ctt gca aag gag aag tcg cag gag aagatg 1168 Phe Lys Glu Ala Ala Arg Leu Ala Lys Glu Lys Ser Gln Glu Lys Met110 115 120 gag ctg acc agt acc cct tca cag gaa tca gca gga gga gat cttcag 1216 Glu Leu Thr Ser Thr Pro Ser Gln Glu Ser Ala Gly Gly Asp Leu Gln125 130 135 tct cct ttg aca cca gaa agt atc aat ggg aca gac gat gag agaaca 1264 Ser Pro Leu Thr Pro Glu Ser Ile Asn Gly Thr Asp Asp Glu Arg Thr140 145 150 ccc gat gtg aca cag aac tca gag cca agg gct gag cca act cagaat 1312 Pro Asp Val Thr Gln Asn Ser Glu Pro Arg Ala Glu Pro Thr Gln Asn155 160 165 gca ttg cca ttt cca cat agg tac aca ttc aat tca gca atc atgatt 1360 Ala Leu Pro Phe Pro His Arg Tyr Thr Phe Asn Ser Ala Ile Met Ile170 175 180 185 aag taaggtggat aaatatggaa gttcatttgg tttcagaaactcttgaagtt 1413 Lys acaacctttg agtgaaaaat ctcaggtcag actcctttaatttattgttc ttggttgctc 1473 aagttgactg aattactata tttccattat ctatgtggaaaaaggagcat tgagctaatt 1533 ataggagaaa ttttttaaat ggagaaaata taattcctttctatctatat tttaaagatc 1593 ccttttgtta acccgttttc tgtntttata tatgttatgtaagatttata atgtgtaatt 1653 agaaacatag aatttctact ctgaaggaaa gctttaccacaggcctacag agttttcaca 1713 gaagacaggg taccaagcac gagcctgtta gcattgatggcagatgccag cagaaggaag 1773 gcttgacttc ctaattctgt attctaaaag atacatcatgttctaaatgc atttcaaaca 1833 ttagttattg gccgtaccgt ggcattactg gactgtaaacatgaatgtga aatggcacta 1893 ttgaaaatat ttttttaaag cccatctacc ttaacactaatttttaccct tatttaaatg 1953 ctttttacta aatagtttta ggtaaaatta agaaaataggggttttttga ctgcacattt 2013 ttttgaagaa ccaagtttta gaaaattata ttctttgacagattaaaaat tgcaaagtga 2073 gatatttcaa actctcctag gtgagttttt attgtgtttgaacttgcatt aataggggca 2133 taggat 2139 <210> SEQ ID NO 24 <211> LENGTH:186 <212> TYPE: PRT <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(2139) <223> OTHERINFORMATION: n is either a, c, g, or t <400> SEQUENCE: 24 Met Gly GluGln Pro Ile Phe Ser Thr Arg Ala His Val Phe Gln Ile 1 5 10 15 Asp ProAsn Thr Lys Lys Asn Trp Val Pro Thr Ser Lys His Ala Val 20 25 30 Thr ValSer Tyr Phe Tyr Asp Ser Thr Arg Asn Val Tyr Arg Ile Ile 35 40 45 Ser LeuAsp Gly Ser Lys Ala Ile Ile Asn Ser Thr Ile Thr Pro Asn 50 55 60 Met ThrPhe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser 65 70 75 80 ArgAla Asn Thr Val Tyr Gly Leu Gly Phe Ser Ser Glu His His Leu 85 90 95 SerLys Phe Ala Glu Lys Phe Gln Glu Phe Lys Glu Ala Ala Arg Leu 100 105 110Ala Lys Glu Lys Ser Gln Glu Lys Met Glu Leu Thr Ser Thr Pro Ser 115 120125 Gln Glu Ser Ala Gly Gly Asp Leu Gln Ser Pro Leu Thr Pro Glu Ser 130135 140 Ile Asn Gly Thr Asp Asp Glu Arg Thr Pro Asp Val Thr Gln Asn Ser145 150 155 160 Glu Pro Arg Ala Glu Pro Thr Gln Asn Ala Leu Pro Phe ProHis Arg 165 170 175 Tyr Thr Phe Asn Ser Ala Ile Met Ile Lys 180 185<210> SEQ ID NO 25 <211> LENGTH: 1418 <212> TYPE: DNA <213> ORGANISM:Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(153)..(1214) <400> SEQUENCE: 25 gaattcggca cgagtctgcc ttgttgagttgcctccggaa aagatctcgg gggtggaaaa 60 gcaactgcaa aataacagac ggagaaaattccttggaagt tatttctgta gcataagagc 120 agaaacttca gagcaagttt tcattgggca aaatg ggg gag caa cct atc ttc 173 Met Gly Glu Gln Pro Ile Phe 1 5 agc actcga gct cat gtc ttc cag att gac ccg aac aca aag aag aac 221 Ser Thr ArgAla His Val Phe Gln Ile Asp Pro Asn Thr Lys Lys Asn 10 15 20 tgg gta cccacc agc aag cat gca gtt act gta tct tat ttt tat gac 269 Trp Val Pro ThrSer Lys His Ala Val Thr Val Ser Tyr Phe Tyr Asp 25 30 35 agc aca aga aatgtg tat agg ata atc agt tta gat ggc tca aag gca 317 Ser Thr Arg Asn ValTyr Arg Ile Ile Ser Leu Asp Gly Ser Lys Ala 40 45 50 55 ata ata aat agcacc atc aca cca aac atg aca ttt act aaa aca tct 365 Ile Ile Asn Ser ThrIle Thr Pro Asn Met Thr Phe Thr Lys Thr Ser 60 65 70 caa aag ttt ggc caatgg gct gat agc cgg gca aac act gtt tat gga 413 Gln Lys Phe Gly Gln TrpAla Asp Ser Arg Ala Asn Thr Val Tyr Gly 75 80 85 ctg gga ttc tcc tct gagcat cat ctt tca aaa ttc gca gaa aag ttt 461 Leu Gly Phe Ser Ser Glu HisHis Leu Ser Lys Phe Ala Glu Lys Phe 90 95 100 cag gaa ttt aag gaa gctgct cgg ctt gca aag gag aag tcg cag gag 509 Gln Glu Phe Lys Glu Ala AlaArg Leu Ala Lys Glu Lys Ser Gln Glu 105 110 115 aag atg gag ctg acc agtacc cct tca cag gaa tca gca gga gga gat 557 Lys Met Glu Leu Thr Ser ThrPro Ser Gln Glu Ser Ala Gly Gly Asp 120 125 130 135 ctt cag tct cct ttgaca cca gaa agt atc aat ggg aca gac gat gag 605 Leu Gln Ser Pro Leu ThrPro Glu Ser Ile Asn Gly Thr Asp Asp Glu 140 145 150 aga aca ccc gat gtgaca cag aac tca gag cca agg gct gag cca act 653 Arg Thr Pro Asp Val ThrGln Asn Ser Glu Pro Arg Ala Glu Pro Thr 155 160 165 cag aat gca ttg ccattt cca cat agt tca gca atc agc aaa cac tgg 701 Gln Asn Ala Leu Pro PhePro His Ser Ser Ala Ile Ser Lys His Trp 170 175 180 gag gct gag cta gctacc ctc aaa ggc aac aat gcc aaa ctc act gca 749 Glu Ala Glu Leu Ala ThrLeu Lys Gly Asn Asn Ala Lys Leu Thr Ala 185 190 195 gcc ctg ctg gag tccact gcc aat gtg aag cag tgg aag caa cag ctt 797 Ala Leu Leu Glu Ser ThrAla Asn Val Lys Gln Trp Lys Gln Gln Leu 200 205 210 215 gct gcg tac caggag gaa gca gag cgg ctg cac aag cgg gtc act gag 845 Ala Ala Tyr Gln GluGlu Ala Glu Arg Leu His Lys Arg Val Thr Glu 220 225 230 ctg gag tgt gttagt agt caa gca aac gct gtg cac agc cac aag aca 893 Leu Glu Cys Val SerSer Gln Ala Asn Ala Val His Ser His Lys Thr 235 240 245 gag ctg aac cagaca gtg cag gaa ctg gaa gag acc ctg aaa gta aag 941 Glu Leu Asn Gln ThrVal Gln Glu Leu Glu Glu Thr Leu Lys Val Lys 250 255 260 gaa gag gaa atagaa aga tta aaa caa gaa atc gat aat gcc aga gaa 989 Glu Glu Glu Ile GluArg Leu Lys Gln Glu Ile Asp Asn Ala Arg Glu 265 270 275 ctc caa gaa cagagg gac tct ttg act cag aaa cta cag gaa gtt gaa 1037 Leu Gln Glu Gln ArgAsp Ser Leu Thr Gln Lys Leu Gln Glu Val Glu 280 285 290 295 att cga aataaa gac ctg gag ggg cag ctg tct gac cta gaa cag cgc 1085 Ile Arg Asn LysAsp Leu Glu Gly Gln Leu Ser Asp Leu Glu Gln Arg 300 305 310 ctg gag aagagc cag aac gaa caa gag gct ttc cgc agt aac ctg aag 1133 Leu Glu Lys SerGln Asn Glu Gln Glu Ala Phe Arg Ser Asn Leu Lys 315 320 325 aca ctc ctagaa att ctg gat gga aaa ata ttt gaa cta aca gaa tta 1181 Thr Leu Leu GluIle Leu Asp Gly Lys Ile Phe Glu Leu Thr Glu Leu 330 335 340 cga gat aatttg gcc aag cta ctg gaa tgc agc taaagagagt gaaatttcag 1234 Arg Asp AsnLeu Ala Lys Leu Leu Glu Cys Ser 345 350 tgccaataga tggagagatg ctgtctgtcttcctaggact gtttgggctc cgtaccaaga 1294 ttgcacaaaa ttttttgaat atcattcctccaggaggagg gtgttttgaa aattggaatt 1354 gtatatttca gtataaattt ttgaatttagcttatagcta attgggaaaa aaaaaaaaaa 1414 aaaa 1418 <210> SEQ ID NO 26 <211>LENGTH: 354 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:26 Met Gly Glu Gln Pro Ile Phe Ser Thr Arg Ala His Val Phe Gln Ile 1 510 15 Asp Pro Asn Thr Lys Lys Asn Trp Val Pro Thr Ser Lys His Ala Val 2025 30 Thr Val Ser Tyr Phe Tyr Asp Ser Thr Arg Asn Val Tyr Arg Ile Ile 3540 45 Ser Leu Asp Gly Ser Lys Ala Ile Ile Asn Ser Thr Ile Thr Pro Asn 5055 60 Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser 6570 75 80 Arg Ala Asn Thr Val Tyr Gly Leu Gly Phe Ser Ser Glu His His Leu85 90 95 Ser Lys Phe Ala Glu Lys Phe Gln Glu Phe Lys Glu Ala Ala Arg Leu100 105 110 Ala Lys Glu Lys Ser Gln Glu Lys Met Glu Leu Thr Ser Thr ProSer 115 120 125 Gln Glu Ser Ala Gly Gly Asp Leu Gln Ser Pro Leu Thr ProGlu Ser 130 135 140 Ile Asn Gly Thr Asp Asp Glu Arg Thr Pro Asp Val ThrGln Asn Ser 145 150 155 160 Glu Pro Arg Ala Glu Pro Thr Gln Asn Ala LeuPro Phe Pro His Ser 165 170 175 Ser Ala Ile Ser Lys His Trp Glu Ala GluLeu Ala Thr Leu Lys Gly 180 185 190 Asn Asn Ala Lys Leu Thr Ala Ala LeuLeu Glu Ser Thr Ala Asn Val 195 200 205 Lys Gln Trp Lys Gln Gln Leu AlaAla Tyr Gln Glu Glu Ala Glu Arg 210 215 220 Leu His Lys Arg Val Thr GluLeu Glu Cys Val Ser Ser Gln Ala Asn 225 230 235 240 Ala Val His Ser HisLys Thr Glu Leu Asn Gln Thr Val Gln Glu Leu 245 250 255 Glu Glu Thr LeuLys Val Lys Glu Glu Glu Ile Glu Arg Leu Lys Gln 260 265 270 Glu Ile AspAsn Ala Arg Glu Leu Gln Glu Gln Arg Asp Ser Leu Thr 275 280 285 Gln LysLeu Gln Glu Val Glu Ile Arg Asn Lys Asp Leu Glu Gly Gln 290 295 300 LeuSer Asp Leu Glu Gln Arg Leu Glu Lys Ser Gln Asn Glu Gln Glu 305 310 315320 Ala Phe Arg Ser Asn Leu Lys Thr Leu Leu Glu Ile Leu Asp Gly Lys 325330 335 Ile Phe Glu Leu Thr Glu Leu Arg Asp Asn Leu Ala Lys Leu Leu Glu340 345 350 Cys Ser <210> SEQ ID NO 27 <211> LENGTH: 1640 <212> TYPE:DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (68)..(1105) <400> SEQUENCE: 27 ggcttggcca cgcgtcgactagtacggggg ggggggcgtc ggagcggccg cacgagcagc 60 gccggag atg gga gaa cagccc atc ttc acc acg cga gcg cac gtc ttc 109 Met Gly Glu Gln Pro Ile PheThr Thr Arg Ala His Val Phe 1 5 10 cag att gac ccc agc acc aag aag aactgg gtg ccg gca agc aag cag 157 Gln Ile Asp Pro Ser Thr Lys Lys Asn TrpVal Pro Ala Ser Lys Gln 15 20 25 30 gcc gtc acg gtt tcc tac ttc tat gatgtc acc agg aac agc tat cgg 205 Ala Val Thr Val Ser Tyr Phe Tyr Asp ValThr Arg Asn Ser Tyr Arg 35 40 45 atc atc agt gtg gat gga gcc aag gtg atcata aac agc act atc acc 253 Ile Ile Ser Val Asp Gly Ala Lys Val Ile IleAsn Ser Thr Ile Thr 50 55 60 ccg aac atg act ttc acc aaa acg tca cag aagttc ggg cag tgg gct 301 Pro Asn Met Thr Phe Thr Lys Thr Ser Gln Lys PheGly Gln Trp Ala 65 70 75 gac agc aga gcc aac acc gtg ttc ggt ttg gga ttctcc tcc gag ctg 349 Asp Ser Arg Ala Asn Thr Val Phe Gly Leu Gly Phe SerSer Glu Leu 80 85 90 cag ctc acg aag ttt gca gag aag ttc cag gag gta agagaa gct gcc 397 Gln Leu Thr Lys Phe Ala Glu Lys Phe Gln Glu Val Arg GluAla Ala 95 100 105 110 agg cta gcc aga gac aag tcc cag gag aaa acc gagacc tcc agc aat 445 Arg Leu Ala Arg Asp Lys Ser Gln Glu Lys Thr Glu ThrSer Ser Asn 115 120 125 cat tcc caa gca tcc agc gtc aat ggc aca gac gacgaa aag gcc tct 493 His Ser Gln Ala Ser Ser Val Asn Gly Thr Asp Asp GluLys Ala Ser 130 135 140 cac gcg agc cca gcc gac act cac ctc aag tct gagaat gac aag ctg 541 His Ala Ser Pro Ala Asp Thr His Leu Lys Ser Glu AsnAsp Lys Leu 145 150 155 aag atc gcg ctg aca cag agt gct gcc aat gtg aagaag tgg gag atg 589 Lys Ile Ala Leu Thr Gln Ser Ala Ala Asn Val Lys LysTrp Glu Met 160 165 170 gag ctg cag acc ctg cgg gag agc aac gcc cgg ctgacc acg gca ctg 637 Glu Leu Gln Thr Leu Arg Glu Ser Asn Ala Arg Leu ThrThr Ala Leu 175 180 185 190 cag gag tcg gcg gcc agc gtg gag cag tgg aagcgg cag ttc tcc atc 685 Gln Glu Ser Ala Ala Ser Val Glu Gln Trp Lys ArgGln Phe Ser Ile 195 200 205 tgc agg gac gag aat gac agg ctc cgc agc aagatc gag gag ctg gaa 733 Cys Arg Asp Glu Asn Asp Arg Leu Arg Ser Lys IleGlu Glu Leu Glu 210 215 220 gaa cag tgc agc gag ata aac agg gag aag gagaag aac aca cag ctg 781 Glu Gln Cys Ser Glu Ile Asn Arg Glu Lys Glu LysAsn Thr Gln Leu 225 230 235 aag agg agg atc gag gag ctg gag tca gag gtccga gac aag gag atg 829 Lys Arg Arg Ile Glu Glu Leu Glu Ser Glu Val ArgAsp Lys Glu Met 240 245 250 gag ttg aaa gat ctc cga aaa cag agt gaa atcata cct cag ctc atg 877 Glu Leu Lys Asp Leu Arg Lys Gln Ser Glu Ile IlePro Gln Leu Met 255 260 265 270 tcc gag tgt gaa tat gtc tct gag aag ttagag gcg gcc gaa aga gac 925 Ser Glu Cys Glu Tyr Val Ser Glu Lys Leu GluAla Ala Glu Arg Asp 275 280 285 aat caa aac ttg gaa gac aaa gtg cgg tctcta aag aca gac atc gag 973 Asn Gln Asn Leu Glu Asp Lys Val Arg Ser LeuLys Thr Asp Ile Glu 290 295 300 gag agt aaa tac cga cag cgc cac ctg aagggg gag ctg aag agc ttc 1021 Glu Ser Lys Tyr Arg Gln Arg His Leu Lys GlyGlu Leu Lys Ser Phe 305 310 315 ctt gag gtg ctg gat gga aag atc gac gacctc cat gac ttc cgt aga 1069 Leu Glu Val Leu Asp Gly Lys Ile Asp Asp LeuHis Asp Phe Arg Arg 320 325 330 gga ctc tcc aag tta ggc aca gat aac tagggc ggg gcggagcaag 1115 Gly Leu Ser Lys Leu Gly Thr Asp Asn Gly Gly 335340 345 tgtgtgtgag aggtgtggta gacgtaggac attctccatt tgcttctgtaaatgcaggtg 1175 cgatctgtct gtctccagac caattgtgcc gtccgctcac tcctccagaataggaaatct 1235 ctcgcttctc tggctttgtg aggtcatgga cagctggaag cttctgactcaggaatccag 1295 aacttggtct accttagccg tttacgcagt cagggcaggg atgtttagatcttcccttaa 1355 gggctgttgt aaccctatga accggggatg ggggagtatt ttctaatccaagtaccatta 1415 tcctttacag caggccctcg ggtgccttct gctgcgtggc attcagtgtatgtgactctc 1475 cagcaggttc tagaccacgg gcatgtggag ggagcatctt ttcccagtatgcattttgtt 1535 gctttagcag atgtgacatg acattgtcaa ccacaaagtt cacactcaaaaactgcacaa 1595 ctgacttact caaaaagaaa taattgtaaa aaaaaaaaaa aaaaa 1640<210> SEQ ID NO 28 <211> LENGTH: 343 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 28 Met Gly Glu Gln Pro Ile Phe Thr Thr Arg AlaHis Val Phe Gln Ile 1 5 10 15 Asp Pro Ser Thr Lys Lys Asn Trp Val ProAla Ser Lys Gln Ala Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Val Thr ArgAsn Ser Tyr Arg Ile Ile 35 40 45 Ser Val Asp Gly Ala Lys Val Ile Ile AsnSer Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln Lys PheGly Gln Trp Ala Asp Ser 65 70 75 80 Arg Ala Asn Thr Val Phe Gly Leu GlyPhe Ser Ser Glu Leu Gln Leu 85 90 95 Thr Lys Phe Ala Glu Lys Phe Gln GluVal Arg Glu Ala Ala Arg Leu 100 105 110 Ala Arg Asp Lys Ser Gln Glu LysThr Glu Thr Ser Ser Asn His Ser 115 120 125 Gln Ala Ser Ser Val Asn GlyThr Asp Asp Glu Lys Ala Ser His Ala 130 135 140 Ser Pro Ala Asp Thr HisLeu Lys Ser Glu Asn Asp Lys Leu Lys Ile 145 150 155 160 Ala Leu Thr GlnSer Ala Ala Asn Val Lys Lys Trp Glu Met Glu Leu 165 170 175 Gln Thr LeuArg Glu Ser Asn Ala Arg Leu Thr Thr Ala Leu Gln Glu 180 185 190 Ser AlaAla Ser Val Glu Gln Trp Lys Arg Gln Phe Ser Ile Cys Arg 195 200 205 AspGlu Asn Asp Arg Leu Arg Ser Lys Ile Glu Glu Leu Glu Glu Gln 210 215 220Cys Ser Glu Ile Asn Arg Glu Lys Glu Lys Asn Thr Gln Leu Lys Arg 225 230235 240 Arg Ile Glu Glu Leu Glu Ser Glu Val Arg Asp Lys Glu Met Glu Leu245 250 255 Lys Asp Leu Arg Lys Gln Ser Glu Ile Ile Pro Gln Leu Met SerGlu 260 265 270 Cys Glu Tyr Val Ser Glu Lys Leu Glu Ala Ala Glu Arg AspAsn Gln 275 280 285 Asn Leu Glu Asp Lys Val Arg Ser Leu Lys Thr Asp IleGlu Glu Ser 290 295 300 Lys Tyr Arg Gln Arg His Leu Lys Gly Glu Leu LysSer Phe Leu Glu 305 310 315 320 Val Leu Asp Gly Lys Ile Asp Asp Leu HisAsp Phe Arg Arg Gly Leu 325 330 335 Ser Lys Leu Gly Thr Asp Asn 340<210> SEQ ID NO 29 <211> LENGTH: 1673 <212> TYPE: DNA <213> ORGANISM:Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(68)..(1129) <400> SEQUENCE: 29 ggcttggcca cgcgtcgact agtacgggggggggggcgtc ggagcggccg cacgagcagc 60 gccggag atg gga gaa cag ccc atc ttcacc acg cga gcg cac gtc ttc 109 Met Gly Glu Gln Pro Ile Phe Thr Thr ArgAla His Val Phe 1 5 10 cag att gac ccc agc acc aag aag aac tgg gtg ccggca agc aag cag 157 Gln Ile Asp Pro Ser Thr Lys Lys Asn Trp Val Pro AlaSer Lys Gln 15 20 25 30 gcc gtc acg gtt tcc tac ttc tat gat gtc acc aggaac agc tat cgg 205 Ala Val Thr Val Ser Tyr Phe Tyr Asp Val Thr Arg AsnSer Tyr Arg 35 40 45 atc atc agt gtg gat gga gcc aag gtg atc ata aac agcact atc acc 253 Ile Ile Ser Val Asp Gly Ala Lys Val Ile Ile Asn Ser ThrIle Thr 50 55 60 ccg aac atg act ttc acc aaa acg tca cag aag ttc ggg cagtgg gct 301 Pro Asn Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln TrpAla 65 70 75 gac agc aga gcc aac acc gtg ttc ggt ttg gga ttc tcc tcc gagctg 349 Asp Ser Arg Ala Asn Thr Val Phe Gly Leu Gly Phe Ser Ser Glu Leu80 85 90 cag ctc acg aag ttt gca gag aag ttc cag gag gta aga gaa gct gcc397 Gln Leu Thr Lys Phe Ala Glu Lys Phe Gln Glu Val Arg Glu Ala Ala 95100 105 110 agg cta gcc aga gac aag tcc cag gag aaa acc gag acc tcc agcaat 445 Arg Leu Ala Arg Asp Lys Ser Gln Glu Lys Thr Glu Thr Ser Ser Asn115 120 125 cat tcc caa gaa tct ggg tgt gaa acc ccg tct tcc act cag gcatcc 493 His Ser Gln Glu Ser Gly Cys Glu Thr Pro Ser Ser Thr Gln Ala Ser130 135 140 agc gtc aat ggc aca gac gac gaa aag gcc tct cac gcg agc ccagcc 541 Ser Val Asn Gly Thr Asp Asp Glu Lys Ala Ser His Ala Ser Pro Ala145 150 155 gac act cac ctc aag tct gag aat gac aag ctg aag atc gcg ctgaca 589 Asp Thr His Leu Lys Ser Glu Asn Asp Lys Leu Lys Ile Ala Leu Thr160 165 170 cag agt gct gcc aat gtg aag aag tgg gag atg gag ctg cag accctg 637 Gln Ser Ala Ala Asn Val Lys Lys Trp Glu Met Glu Leu Gln Thr Leu175 180 185 190 cgg gag agc aac gcc cgg ctg acc acg gca ctg cag gag tcggcg gcc 685 Arg Glu Ser Asn Ala Arg Leu Thr Thr Ala Leu Gln Glu Ser AlaAla 195 200 205 agc gtg gag cag tgg aag cgg cag ttc tcc atc tgc agg gacgag aat 733 Ser Val Glu Gln Trp Lys Arg Gln Phe Ser Ile Cys Arg Asp GluAsn 210 215 220 gac agg ctc cgc agc aag atc gag gag ctg gaa gaa cag tgcagc gag 781 Asp Arg Leu Arg Ser Lys Ile Glu Glu Leu Glu Glu Gln Cys SerGlu 225 230 235 ata aac agg gag aag gag aag aac aca cag ctg aag agg aggatc gag 829 Ile Asn Arg Glu Lys Glu Lys Asn Thr Gln Leu Lys Arg Arg IleGlu 240 245 250 gag ctg gag tca gag gtc cga gac aag gag atg gag ttg aaagat ctc 877 Glu Leu Glu Ser Glu Val Arg Asp Lys Glu Met Glu Leu Lys AspLeu 255 260 265 270 cga aaa cag agt gaa atc ata cct cag ctc atg tcc gagtgt gaa tat 925 Arg Lys Gln Ser Glu Ile Ile Pro Gln Leu Met Ser Glu CysGlu Tyr 275 280 285 gtc tct gag aag tta gag gcg gcc gaa aga gac aat caaaac ttg gaa 973 Val Ser Glu Lys Leu Glu Ala Ala Glu Arg Asp Asn Gln AsnLeu Glu 290 295 300 gac aaa gtg cgg tct cta aag aca gac atc gag gag agtaaa tac cga 1021 Asp Lys Val Arg Ser Leu Lys Thr Asp Ile Glu Glu Ser LysTyr Arg 305 310 315 cag cgc cac ctg aag ggg gag ctg aag agc ttc ctt gaggtg ctg gat 1069 Gln Arg His Leu Lys Gly Glu Leu Lys Ser Phe Leu Glu ValLeu Asp 320 325 330 gga aag atc gac gac ctc cat gac ttc cgt aga gga ctctcc aag tta 1117 Gly Lys Ile Asp Asp Leu His Asp Phe Arg Arg Gly Leu SerLys Leu 335 340 345 350 ggc aca gat aac tagggcgggg cggagcaagt gtgtgtgagaggtgtggtag 1169 Gly Thr Asp Asn acgtaggaca ttctccattt gcttctgtaaatgcaggtgc gatctgtctg tctccagacc 1229 aattgtgccg tccgctcact cctccagaataggaaatctc tcgcttctct ggctttgtga 1289 ggtcatggac agctggaagc ttctgactcaggaatccaga acttggtcta ccttagccgt 1349 ttacgcagtc agggcaggga tgtttagatcttcccttaag ggctgttgta accctatgaa 1409 ccggggatgg gggagtattt tctaatccaagtaccattat cctttacagc aggccctcgg 1469 gtgccttctg ctgcgtggca ttcagtgtatgtgactctcc agcaggttct agaccacggg 1529 catgtggagg gagcatcttt tcccagtatgcattttgttg ctttagcaga tgtgacatga 1589 cattgtcaac cacaaagttc acactcaaaaactgcacaac tgacttactc aaaaagaaat 1649 aattgtaaaa aaaaaaaaaa aaaa 1673<210> SEQ ID NO 30 <211> LENGTH: 354 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 30 Met Gly Glu Gln Pro Ile Phe Thr Thr Arg AlaHis Val Phe Gln Ile 1 5 10 15 Asp Pro Ser Thr Lys Lys Asn Trp Val ProAla Ser Lys Gln Ala Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Val Thr ArgAsn Ser Tyr Arg Ile Ile 35 40 45 Ser Val Asp Gly Ala Lys Val Ile Ile AsnSer Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln Lys PheGly Gln Trp Ala Asp Ser 65 70 75 80 Arg Ala Asn Thr Val Phe Gly Leu GlyPhe Ser Ser Glu Leu Gln Leu 85 90 95 Thr Lys Phe Ala Glu Lys Phe Gln GluVal Arg Glu Ala Ala Arg Leu 100 105 110 Ala Arg Asp Lys Ser Gln Glu LysThr Glu Thr Ser Ser Asn His Ser 115 120 125 Gln Glu Ser Gly Cys Glu ThrPro Ser Ser Thr Gln Ala Ser Ser Val 130 135 140 Asn Gly Thr Asp Asp GluLys Ala Ser His Ala Ser Pro Ala Asp Thr 145 150 155 160 His Leu Lys SerGlu Asn Asp Lys Leu Lys Ile Ala Leu Thr Gln Ser 165 170 175 Ala Ala AsnVal Lys Lys Trp Glu Met Glu Leu Gln Thr Leu Arg Glu 180 185 190 Ser AsnAla Arg Leu Thr Thr Ala Leu Gln Glu Ser Ala Ala Ser Val 195 200 205 GluGln Trp Lys Arg Gln Phe Ser Ile Cys Arg Asp Glu Asn Asp Arg 210 215 220Leu Arg Ser Lys Ile Glu Glu Leu Glu Glu Gln Cys Ser Glu Ile Asn 225 230235 240 Arg Glu Lys Glu Lys Asn Thr Gln Leu Lys Arg Arg Ile Glu Glu Leu245 250 255 Glu Ser Glu Val Arg Asp Lys Glu Met Glu Leu Lys Asp Leu ArgLys 260 265 270 Gln Ser Glu Ile Ile Pro Gln Leu Met Ser Glu Cys Glu TyrVal Ser 275 280 285 Glu Lys Leu Glu Ala Ala Glu Arg Asp Asn Gln Asn LeuGlu Asp Lys 290 295 300 Val Arg Ser Leu Lys Thr Asp Ile Glu Glu Ser LysTyr Arg Gln Arg 305 310 315 320 His Leu Lys Gly Glu Leu Lys Ser Phe LeuGlu Val Leu Asp Gly Lys 325 330 335 Ile Asp Asp Leu His Asp Phe Arg ArgGly Leu Ser Lys Leu Gly Thr 340 345 350 Asp Asn <210> SEQ ID NO 31 <211>LENGTH: 2297 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1023) <400> SEQUENCE: 31 tccaca gcc agg gaa cag cca atc ttc agc acc cgg gcg cac gta ttc 48 Ser ThrAla Arg Glu Gln Pro Ile Phe Ser Thr Arg Ala His Val Phe 1 5 10 15 cagatc gac ccc act aca aag cgg aac tgg atc ccc gcc ggc aag cac 96 Gln IleAsp Pro Thr Thr Lys Arg Asn Trp Ile Pro Ala Gly Lys His 20 25 30 gca cttacc gtg tcc tat ttc tat gat gca acc cga aat gtg tac cgc 144 Ala Leu ThrVal Ser Tyr Phe Tyr Asp Ala Thr Arg Asn Val Tyr Arg 35 40 45 atc atc agcatc ggg ggt gcc aag gcc atc atc aac agc act gtc act 192 Ile Ile Ser IleGly Gly Ala Lys Ala Ile Ile Asn Ser Thr Val Thr 50 55 60 ccc aac atg accttc acc aaa acc tct cag aag ttc ggg caa tgg gca 240 Pro Asn Met Thr PheThr Lys Thr Ser Gln Lys Phe Gly Gln Trp Ala 65 70 75 80 gac agt cga gccaac act gtc tac ggc cta ggc ttt gcc tct gaa cag 288 Asp Ser Arg Ala AsnThr Val Tyr Gly Leu Gly Phe Ala Ser Glu Gln 85 90 95 cag ctg acc cag tttgct gag aag ttt cag gag gtg aaa gaa gct gcc 336 Gln Leu Thr Gln Phe AlaGlu Lys Phe Gln Glu Val Lys Glu Ala Ala 100 105 110 agg ctg gct cga gagaaa tct caa gat ggt gga gaa ttc act agt act 384 Arg Leu Ala Arg Glu LysSer Gln Asp Gly Gly Glu Phe Thr Ser Thr 115 120 125 ggc ctg gcc ctt gcctcc cat cag gtt cct cca agc ccc ttg gtc agc 432 Gly Leu Ala Leu Ala SerHis Gln Val Pro Pro Ser Pro Leu Val Ser 130 135 140 acc aat ggt cca ggcgag gaa aag ctg ttc cgt agc cag agt gcg gac 480 Thr Asn Gly Pro Gly GluGlu Lys Leu Phe Arg Ser Gln Ser Ala Asp 145 150 155 160 acc cct ggc cccacc gag cgg gaa cgg ttg aag aag atg ctg tca gaa 528 Thr Pro Gly Pro ThrGlu Arg Glu Arg Leu Lys Lys Met Leu Ser Glu 165 170 175 ggc tct gta ggggaa gtc cag tgg gaa gca gag ttc ttc gcg ctt cag 576 Gly Ser Val Gly GluVal Gln Trp Glu Ala Glu Phe Phe Ala Leu Gln 180 185 190 gac agc aac cagagg ttg gcg gga gcc ctt cgg gaa gcg aac gca gcg 624 Asp Ser Asn Gln ArgLeu Ala Gly Ala Leu Arg Glu Ala Asn Ala Ala 195 200 205 gcc act cag tggagg caa caa ctg gag gtc caa cgt gca gag gct gaa 672 Ala Thr Gln Trp ArgGln Gln Leu Glu Val Gln Arg Ala Glu Ala Glu 210 215 220 ctc ttg agg cagcgg gta gca gag ctg gag gcc cag gtg gct gta gag 720 Leu Leu Arg Gln ArgVal Ala Glu Leu Glu Ala Gln Val Ala Val Glu 225 230 235 240 cca gtc cgggca gga gag aaa gaa gca acc agc cag tcg gtg gag cag 768 Pro Val Arg AlaGly Glu Lys Glu Ala Thr Ser Gln Ser Val Glu Gln 245 250 255 ctg gag gctcgg gtg cag acc aag gac cag gag atc cag act ttg aag 816 Leu Glu Ala ArgVal Gln Thr Lys Asp Gln Glu Ile Gln Thr Leu Lys 260 265 270 aat cag agcact ggc acc cga gag gct cca gac act gcc gag cgc gaa 864 Asn Gln Ser ThrGly Thr Arg Glu Ala Pro Asp Thr Ala Glu Arg Glu 275 280 285 gag aca cagcag caa gtt cag gac ctg gag acc cgg aat gca gag ctg 912 Glu Thr Gln GlnGln Val Gln Asp Leu Glu Thr Arg Asn Ala Glu Leu 290 295 300 gag cag cagctg cgg gcg atg gag tgc aac ctg gag gag gcg cgg gcc 960 Glu Gln Gln LeuArg Ala Met Glu Cys Asn Leu Glu Glu Ala Arg Ala 305 310 315 320 gag cgggag cgc gca cgg gcg gag gtg ggc cgg gct gcg cag ctg ctg 1008 Glu Arg GluArg Ala Arg Ala Glu Val Gly Arg Ala Ala Gln Leu Leu 325 330 335 gat gttcgg ctg ttt gagctcagcg agctgcgtga aggcctggca cgcctggcag 1063 Asp Val ArgLeu Phe 340 aggcagcacc ctagtctgcc atggagtgtc tgcggcctca aggcgccctggcaggggcca 1123 ggggacccca gctgtctctg agctttgcac tgtgtagagt tttctagaatccttgggcaa 1183 tgcttctacc caggttacat ttctacgtgt ggcgttgctg tccctggctgctgctgccct 1243 gcgccccagg gacactgcga gggaaggctg cactagtcat ccccatggggcaacagaggc 1303 tttgggatcc tgagacctga aggccctgta ctcatcccac cccattctcaagtcagactg 1363 acaacttcaa agagtgttta ctgaagtcag gggccaccag caccaggtttacagctcagt 1423 cctgagcctc agcctgggct ggctcttggg gccgagatct gggaggacgcgaccgtcgga 1483 cagtgctccc tgctttctgc cgccgaagtg tctgccccac tttctccttgaagcgtcggt 1543 tttgttgctt gatcttggcc agctcagctt tgcgtttggc ctccaggtctgggtcctgcg 1603 gaagggagct gagaatgtaa ctgggcagct tcccagggac tggctcccccacccctaccc 1663 gtccccaggt cccacccacc cttactggcc acactcttat gcctgtccctgcatacccat 1723 gcctccctat actaccttcc cctccaggat catctgtttc cgcttgttgatctctttctt 1783 ttcatcaaaa tgcgaagcct ccagtttcta ggggtgggga ggggaacaggtcagtcaggc 1843 ctggggcagg aagccccgcc cacctcaccc cactccaccc taccctgacaggctggccac 1903 acttactatt tcgcactccc ttcgcactac gttgacctgc gtgaggatttgtagaacctc 1963 agcctcctcc accaccagct ctgccagctg ctgctctgca gggacaggaaacactgagtt 2023 gggctgggag tgcaaccagc cctctgcacc cccagctctg gatgtctggatccaaccaaa 2083 tgtggactga tgatatttag aaaaagcaaa atgctgccaa gcttggcagcacatgcttgt 2143 catcacagca ctgggaggtg gaggcagggg gatcactcgt ttcagctgagttccaggcca 2203 gctctgtaga gcaagaatct gtctcaaatt aatgactgaa taaacaaatgaacaagtaaa 2263 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 2297 <210> SEQ IDNO 32 <211> LENGTH: 341 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 32 Ser Thr Ala Arg Glu Gln Pro Ile Phe Ser Thr Arg AlaHis Val Phe 1 5 10 15 Gln Ile Asp Pro Thr Thr Lys Arg Asn Trp Ile ProAla Gly Lys His 20 25 30 Ala Leu Thr Val Ser Tyr Phe Tyr Asp Ala Thr ArgAsn Val Tyr Arg 35 40 45 Ile Ile Ser Ile Gly Gly Ala Lys Ala Ile Ile AsnSer Thr Val Thr 50 55 60 Pro Asn Met Thr Phe Thr Lys Thr Ser Gln Lys PheGly Gln Trp Ala 65 70 75 80 Asp Ser Arg Ala Asn Thr Val Tyr Gly Leu GlyPhe Ala Ser Glu Gln 85 90 95 Gln Leu Thr Gln Phe Ala Glu Lys Phe Gln GluVal Lys Glu Ala Ala 100 105 110 Arg Leu Ala Arg Glu Lys Ser Gln Asp GlyGly Glu Phe Thr Ser Thr 115 120 125 Gly Leu Ala Leu Ala Ser His Gln ValPro Pro Ser Pro Leu Val Ser 130 135 140 Thr Asn Gly Pro Gly Glu Glu LysLeu Phe Arg Ser Gln Ser Ala Asp 145 150 155 160 Thr Pro Gly Pro Thr GluArg Glu Arg Leu Lys Lys Met Leu Ser Glu 165 170 175 Gly Ser Val Gly GluVal Gln Trp Glu Ala Glu Phe Phe Ala Leu Gln 180 185 190 Asp Ser Asn GlnArg Leu Ala Gly Ala Leu Arg Glu Ala Asn Ala Ala 195 200 205 Ala Thr GlnTrp Arg Gln Gln Leu Glu Val Gln Arg Ala Glu Ala Glu 210 215 220 Leu LeuArg Gln Arg Val Ala Glu Leu Glu Ala Gln Val Ala Val Glu 225 230 235 240Pro Val Arg Ala Gly Glu Lys Glu Ala Thr Ser Gln Ser Val Glu Gln 245 250255 Leu Glu Ala Arg Val Gln Thr Lys Asp Gln Glu Ile Gln Thr Leu Lys 260265 270 Asn Gln Ser Thr Gly Thr Arg Glu Ala Pro Asp Thr Ala Glu Arg Glu275 280 285 Glu Thr Gln Gln Gln Val Gln Asp Leu Glu Thr Arg Asn Ala GluLeu 290 295 300 Glu Gln Gln Leu Arg Ala Met Glu Cys Asn Leu Glu Glu AlaArg Ala 305 310 315 320 Glu Arg Glu Arg Ala Arg Ala Glu Val Gly Arg AlaAla Gln Leu Leu 325 330 335 Asp Val Arg Leu Phe 340 <210> SEQ ID NO 33<211> LENGTH: 5798 <212> TYPE: DNA <213> ORGANISM: Rattus norvegicus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (567)..(1124) <400>SEQUENCE: 33 gtgctgtgca catcgcgagc ggctggggtt tgcacttcga gatttcttctttataatttt 60 ttttttttaa tgtaagggag acagtggaat tgctacccgt agaatttttattcaagtgca 120 cgtcgcgttg ggttgcacgc tccaccccca gggacctggt gtggtgaaatttgaacccac 180 cgccttagcc caaaggccga gtaacctggc tgcttgagtg tcgtggaagacgtgagcgaa 240 atgatcagcg aactcatttt ttatcagact cgctgaagct ggcttttgcgtttttctaca 300 cgtacactaa ttttatggaa tagttaaagt gctatattct ccgcgcaaccttttcaaatt 360 ccaaatgttt gaacgttttg gtgtcagcgc gagtgaaatc attttaccgacaagaactaa 420 ctgaattgtc tgcctcgttg agttgcctcc ggaaaagatc tcgggggtggaaaagcaact 480 gcaaaataac agacggagaa aattccttgg aagttatttc tgtagcataagagcagaaac 540 ttcagagcaa gttttcattg ggcaaa atg ggg gaa caa cct atc ttcagc act 593 Met Gly Glu Gln Pro Ile Phe Ser Thr 1 5 cga gct cat gtc ttccag atc gac cca aac aca aag aag aac tgg gta 641 Arg Ala His Val Phe GlnIle Asp Pro Asn Thr Lys Lys Asn Trp Val 10 15 20 25 ccc acc agc aag catgca gtt act gtg tct tat ttc tat gac agc aca 689 Pro Thr Ser Lys His AlaVal Thr Val Ser Tyr Phe Tyr Asp Ser Thr 30 35 40 agg aat gtg tat agg ataatc agt cta gac ggc tca aag gca ata ata 737 Arg Asn Val Tyr Arg Ile IleSer Leu Asp Gly Ser Lys Ala Ile Ile 45 50 55 aat agc acc atc act cca aacatg aca ttt act aaa aca tct caa aag 785 Asn Ser Thr Ile Thr Pro Asn MetThr Phe Thr Lys Thr Ser Gln Lys 60 65 70 ttt ggc caa tgg gct gat agc cgggca aac act gtt tat gga ctg gga 833 Phe Gly Gln Trp Ala Asp Ser Arg AlaAsn Thr Val Tyr Gly Leu Gly 75 80 85 ttc tcc tct gag cat cat ctc tca aaattt gca gaa aag ttt cag gaa 881 Phe Ser Ser Glu His His Leu Ser Lys PheAla Glu Lys Phe Gln Glu 90 95 100 105 ttt aaa gaa gct gct cgg ctg gcaaag gag aag tcg cag gag aag atg 929 Phe Lys Glu Ala Ala Arg Leu Ala LysGlu Lys Ser Gln Glu Lys Met 110 115 120 gaa ctg acc agt acc cct tca caggaa tca gca gga gga gat ctt cag 977 Glu Leu Thr Ser Thr Pro Ser Gln GluSer Ala Gly Gly Asp Leu Gln 125 130 135 tct cct tta aca cca gaa agt atcaat ggg aca gat gat gag aga aca 1025 Ser Pro Leu Thr Pro Glu Ser Ile AsnGly Thr Asp Asp Glu Arg Thr 140 145 150 ccc gat gtg aca cag aac tca gagcca agg gct gag cca gct cag aat 1073 Pro Asp Val Thr Gln Asn Ser Glu ProArg Ala Glu Pro Ala Gln Asn 155 160 165 gca ttg cca ttt tca cat agg tacaca ttc aat tca gca atc atg att 1121 Ala Leu Pro Phe Ser His Arg Tyr ThrPhe Asn Ser Ala Ile Met Ile 170 175 180 185 aaa tgagatggat aaatatgaagttcatttggt ttcagaaact cttgagtgaa 1174 Lys aaatcccagg tcagacttctttaattaatt aattgtttgc tgttgctcag attgactgaa 1234 tatttccatt atctgtgtagaaaaaggaac gttaattata ggagaaactt tttcaatgga 1294 caaaacattc cattctatctatattttaaa gatccctttt gctaaccagt tttctgattt 1354 tctacatgtt acgtaagactaataacttgt gattaggatc aatggactcc tgctccaaag 1414 gaaagccttg ccacaggcccacagaggtgc cacagaggac ggggccaggc aggaacccgt 1474 cagcattgaa ggttgtttttgtatgccaac aggaggaaag cttgagttgc tgctgattct 1534 taaaagaatt ctgtattctaaaagatacac atcatgttct aaatgcattt taaactagtg 1594 acattagtta ttgggcatactgtggtatta ctagactaca aagaggaata tgaagtggca 1654 ccattgaaag tatttttttaaaaagcctgt ctaccttaac actaattttt acccttattt 1714 aaatgctttt tactaaacagttttaggtaa aattaagaaa acagttttgt tgactgcaca 1774 tcttttagaa ggaccaacttttagagaatt acattctttg acagattaaa aattgcaaag 1834 tgagatattt caaactcttaagtgagtttt attgccgttg gactgcatta atacggacat 1894 acgattaaac ttagtagaccaacactgagg gatctcctta ccaggctgca gaacaaggaa 1954 attaagcaat aaatgggacttgtgaatgga aggacactct actgctagtg ctagtaattc 2014 tgcataagat ggtatacattttgaagaaag ctgcttttaa ttacttttaa taatgatttt 2074 aattactcta gtgcaagtgcttcctcgagc tataaaggta gctgagcaca gcagaccttt 2134 actccctcag tctgacttctgtactcatat tcatttagtg aacatagtct tttaacagaa 2194 gaccacagtt ctttgatagcgttacaaaac ttacgttatt taaacgttat aaagaacgtt 2254 attgtaggat aaaatgttaaaaactgtgtc aaggacagga agaattccta tctattaagt 2314 agtggtttcc acccccacttaagactgaac tgcactgaac ggtaactgta tacttggttt 2374 gacacctcga ctgagccatgcgcactgaat actgtgacat tgaggagtaa gaacttttaa 2434 atttaacatt taaagaagctacttgcagtt tatgcaccga aatttgtcta aatgttctcc 2494 attttgctga ccccgttgtattcatactgc tccccagagc ctagagttgt cctcatcctg 2554 acttcctgtg cctgagtgtctgagaggagt cactttcact gtgaagacac tgcttctgcg 2614 cctcgtaggg aggacttgacagtgctcccg tagaaatcct acattatttc aacctcagag 2674 ttacagtaaa ggcaggttataaccagtctt tcttattatt ttaagaattt ccagccctag 2734 tgttttatga aagtattcctgtgaatttga caccttatga tcctatattc atctaattcc 2794 ttaatgaaat aaaaatgtccatgtgaggta ggttatttac agcgattgca ggagacatgg 2854 tgttcttcag agttcccaaaccaggatagt ttcaaatagg tttttcatgg cttctgacga 2914 agaagaccat aaagttccctgcagtgtgtc agtgatgtgc aagctgaatt agtgcgaagt 2974 gtcacactgt gaaagcacgtgcttttggct tattatgaga aaacgaaatc tttaaattca 3034 gtttatgtgt cttaggtccagtttactttg atttgactac tcagttcttc tgaccccacc 3094 tagtatgtat gtatatgtgtgtgtatgtgt gtgtatgtct gtatgtatat acatacatat 3154 acacacacat tgtatacatatgctatatat acagtatgtg tatatatata ctatatatga 3214 atatatgaat atatatattcaattagttaa tagtacattt aagccaaata tccaacataa 3274 gcacactatg taagtatctatctggaaaga cctatataga attgagatca acatttcatg 3334 agttagaaac aaaggattttataattaata ttacttaagt ctaaagtacc catatattta 3394 aattagatat gcaatttttccctcttggca aagaaagaca aaaatcttgt gtttagagat 3454 gatgtagatt gtcatttttgcctttccttc ctgagtactt gttttaacaa caacaaaaaa 3514 agactagttt aagaaaagggattgtccagt atttttctgc tttgttaagt ctaattttac 3574 tgttaaacag agagcagaatcactggagta ctgggggggt tttttgttgt tttttttttt 3634 ttcttttctg tttttttcggagctggggag cgaacccagg gccttgcgct cactaggcaa 3694 gcactctacc gctgagctaaatccccaacc cctggagtat ctgttttaaa agaaagccag 3754 gaccgttatg atggccatacccagggtaca tagtgaaaac aacagagacc aagcaatgag 3814 agtgtgagag taccaatccaccagtactgc tgccggacat ggcagctgcc tgtgcttttc 3874 tgaagagtca tagtgtatgctaagtctaga accattactt agtaaagagg ctatgacttt 3934 tatttgggcc tgacaattttagtggtgtgg tcatagtcta ttctgtattt gtaagcttta 3994 tttttaaatt actgtgttgatttaggaaca caagaaatgt ttttattttt aattatgagt 4054 gtatataagg ttttcagatatgcacagact acaataatag actcccatgg agataccact 4114 tcagccttaa cagtcagggagaaggagcct cactttatca ccgcactcac cctgctctcc 4174 actgatctgt tgttactgcggtgtggaggt tcacacgcat gcaggtcttc acacatgatg 4234 ggtaggcccg caccaagtgagcctctccca gccttgctgt ttcgtttttt tattttaatc 4294 ttacatgtat gggtgttttgcatccaggca tgtcatgcct gtgtccacag aagccagaaa 4354 gggtatcaga ttccctaaaactggagttct cgatgatcgt gagcgagcca ttgtgggtgc 4414 tgggaactga agctgggtcctctacaagag cagccagcgc tcttaaccat tgagccacta 4474 tctgccctgt gtttgttttatttatttatt tatttattta tttatttatt tatttattta 4534 tttatttatt tatttatttattggttcttt ttttttggac tggggaccga agccagggcc 4594 ttgcacttcc taggcaagcgctctaccact gagctaaatc cccaacccct tgttttattt 4654 ttaaagcaaa cgagatacataatttcaacc atgataattt aagattatct tgaactctta 4714 aggaaatgta tatactaagctattatagtt tttattttcc ctaattcagt ggcataatac 4774 cttaccttga gtcgtttactactttctttg gtttctaaaa actctactgc taaattacaa 4834 tgtaaaaaca tagggctcgtatatactgta gagtgctgta gatgtcctcg tcatcaacta 4894 tgcaataaca gtctgatcgacacatttcag gagcgatcac tctttggtgt gcttctttaa 4954 atactttcag aagcttaggatgtgcaaagc aggaagaccg tgggtgtaaa tgtttactta 5014 tttctttgag agtgttagtaagtcttttct aaattgcttt tctcttcaaa attatcgtta 5074 acttaaatga taattatctttgaggttaaa cagaagctca ttgacaaact aaagtgactt 5134 tttagggcat tctttgagatcatagtctta tatctgggga ctaaaatgtc attagaccct 5194 aatagactaa cttgtatgtttgtgtgggga aacgttttcc tctctcattc aaggtaactg 5254 tttgctgcct gttgttacttgtgtagcatt ctagaaaatg gctaggtttt ttataagatt 5314 taagacaata gaagtagttttatattatta tagttctgtt ggaatgtgat cctgaaatta 5374 ttactgaaaa ttagaatttttatttcgcta atgacaacct tgactctcag agatgcagtg 5434 taaattgata cctcatctttccgagagttc agagcacagg gcggcagtat gtgaagctgc 5494 ttttgcactg acgcattttgataagtttgg ctactgtaat ggtaaaaggc tcctcaggca 5554 ctgactgcat ttgggttcttccgatggggg atgatccgtt ctcgtggtgc tgctggactt 5614 atgcattttg gaggtactgcatgtatcttc cacactgctt gacattttct ctgatctgtg 5674 tgtttgcacc aactcattaaaagaaatatg cagaaatatc ttctaattcg ttgatcttcg 5734 ctgtatgaca gttataatattaaacacttg ggttgatcaa aaaaaaaaaa aaaaaaaaaa 5794 aaaa 5798 <210> SEQ IDNO 34 <211> LENGTH: 186 <212> TYPE: PRT <213> ORGANISM: Rattusnorvegicus <400> SEQUENCE: 34 Met Gly Glu Gln Pro Ile Phe Ser Thr ArgAla His Val Phe Gln Ile 1 5 10 15 Asp Pro Asn Thr Lys Lys Asn Trp ValPro Thr Ser Lys His Ala Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Ser ThrArg Asn Val Tyr Arg Ile Ile 35 40 45 Ser Leu Asp Gly Ser Lys Ala Ile IleAsn Ser Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln LysPhe Gly Gln Trp Ala Asp Ser 65 70 75 80 Arg Ala Asn Thr Val Tyr Gly LeuGly Phe Ser Ser Glu His His Leu 85 90 95 Ser Lys Phe Ala Glu Lys Phe GlnGlu Phe Lys Glu Ala Ala Arg Leu 100 105 110 Ala Lys Glu Lys Ser Gln GluLys Met Glu Leu Thr Ser Thr Pro Ser 115 120 125 Gln Glu Ser Ala Gly GlyAsp Leu Gln Ser Pro Leu Thr Pro Glu Ser 130 135 140 Ile Asn Gly Thr AspAsp Glu Arg Thr Pro Asp Val Thr Gln Asn Ser 145 150 155 160 Glu Pro ArgAla Glu Pro Ala Gln Asn Ala Leu Pro Phe Ser His Arg 165 170 175 Tyr ThrPhe Asn Ser Ala Ile Met Ile Lys 180 185 <210> SEQ ID NO 35 <211> LENGTH:3339 <212> TYPE: DNA <213> ORGANISM: Rattus norvegicus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (740)..(1801) <400> SEQUENCE: 35ctagtggatc ccccgggctg caggaattct gcggccgcaa caccgcactg tggtggacag 60tgagggccgg agagagacca cagtgaccca tcaagaagcc catgacagtt ccagaagtga 120tccagatcct ccaagatctt cagctttgga tgatcccttt tccatcctgg acctgcttct 180aggacgttgg tttcggtccc gatagctttc ttgaacttca gaggccttca ggtccttccc 240accccctccc tccctgttgc ccattgccaa taagcatagc ttttgctgtc atcctggggt 300cttaaatgtg tggaaccccc ccagggacct ggtgtggtga aatttgaacc caccgcctta 360gcccaaaggc cgagtaacct ggctgcttga gtgtcgtgga agacgtgagc gaaatgatca 420gcgaactcat tttttatcag actcgctgaa gctggctttt gcgtttttct acacgtacac 480taattttatg gaatagttaa agtgctatat tctccgcgca accttttcaa attccaaatg 540tttgaacgtt ttggtgtcag cgcgagtgaa atcattttac cgacaagaac taactgaatt 600gtctgcctcg ttgagttgcc tccggaaaag atctcggggg tggaaaagca actgcaaaat 660aacagacgga gaaaattcct tggaagttat ttctgtagca taagagcaga aacttaagag 720caagttttca ttgggcaaa atg ggg gaa caa cct atc ttc agc act cga gct 772 MetGly Glu Gln Pro Ile Phe Ser Thr Arg Ala 1 5 10 cat gtc ttc cag atc gaccca aac aca aag aag aac tgg gta ccc acc 820 His Val Phe Gln Ile Asp ProAsn Thr Lys Lys Asn Trp Val Pro Thr 15 20 25 agc aag cat gca gtt act gtgtct tat ttc tat gac agc aca agg aat 868 Ser Lys His Ala Val Thr Val SerTyr Phe Tyr Asp Ser Thr Arg Asn 30 35 40 gtg tat agg ata atc agt cta gacggc tca aag gca ata ata aat agc 916 Val Tyr Arg Ile Ile Ser Leu Asp GlySer Lys Ala Ile Ile Asn Ser 45 50 55 acc atc act cca aac atg aca ttt actaaa aca tct caa aag ttt ggc 964 Thr Ile Thr Pro Asn Met Thr Phe Thr LysThr Ser Gln Lys Phe Gly 60 65 70 75 caa tgg gct gat agc cgg gca aac actgtt tat gga ctg gga ttc tcc 1012 Gln Trp Ala Asp Ser Arg Ala Asn Thr ValTyr Gly Leu Gly Phe Ser 80 85 90 tct gag cat cat ctc tca aaa ttt gca gaaaag ttt cag gaa ttt aaa 1060 Ser Glu His His Leu Ser Lys Phe Ala Glu LysPhe Gln Glu Phe Lys 95 100 105 gaa gct gct cgg ctg gca aag gag aag tcgcag gag aag atg gaa ctg 1108 Glu Ala Ala Arg Leu Ala Lys Glu Lys Ser GlnGlu Lys Met Glu Leu 110 115 120 acc agt acc cct tca cag gaa tca gca ggagga gat ctt cag tct cct 1156 Thr Ser Thr Pro Ser Gln Glu Ser Ala Gly GlyAsp Leu Gln Ser Pro 125 130 135 tta aca cca gaa agt atc aat ggg aca gatgat gag aga aca ccc gat 1204 Leu Thr Pro Glu Ser Ile Asn Gly Thr Asp AspGlu Arg Thr Pro Asp 140 145 150 155 gtg aca cag aac tca gag cca agg gctgag cca gct cag aat gca ttg 1252 Val Thr Gln Asn Ser Glu Pro Arg Ala GluPro Ala Gln Asn Ala Leu 160 165 170 cca ttt tca cat agt tca gcc atc agcaaa cac tgg gag gct gaa cta 1300 Pro Phe Ser His Ser Ser Ala Ile Ser LysHis Trp Glu Ala Glu Leu 175 180 185 gcc acg ctc aag ggg aac aat gcc aagctc acc gca gcg ctg ctg gag 1348 Ala Thr Leu Lys Gly Asn Asn Ala Lys LeuThr Ala Ala Leu Leu Glu 190 195 200 tcc act gcc aac gtg aag cag tgg aagcaa cag ctg gct gcc tac cag 1396 Ser Thr Ala Asn Val Lys Gln Trp Lys GlnGln Leu Ala Ala Tyr Gln 205 210 215 gag gag gca gag cgg ctg cac aag cgggtc acg gag ctg gaa tgt gtt 1444 Glu Glu Ala Glu Arg Leu His Lys Arg ValThr Glu Leu Glu Cys Val 220 225 230 235 agt agt caa gca aac gcg gtg cacagc cac aag aca gag ctg agt cag 1492 Ser Ser Gln Ala Asn Ala Val His SerHis Lys Thr Glu Leu Ser Gln 240 245 250 aca gtg cag gag ctg gaa gag acccta aaa gta aag gaa gag gaa ata 1540 Thr Val Gln Glu Leu Glu Glu Thr LeuLys Val Lys Glu Glu Glu Ile 255 260 265 gaa aga tta aaa caa gaa att gataac gcc aga gaa ctt caa gaa cag 1588 Glu Arg Leu Lys Gln Glu Ile Asp AsnAla Arg Glu Leu Gln Glu Gln 270 275 280 agg gac tct ttg act cag aaa ctacag gaa gtt gag att cga aat aaa 1636 Arg Asp Ser Leu Thr Gln Lys Leu GlnGlu Val Glu Ile Arg Asn Lys 285 290 295 gac ctg gag ggg cag ctg tcg gagctg gag cag cgc ctg gag aag agc 1684 Asp Leu Glu Gly Gln Leu Ser Glu LeuGlu Gln Arg Leu Glu Lys Ser 300 305 310 315 cag agc gag cag gac gct ttccgc agt aac ctg aag act ctc cta gag 1732 Gln Ser Glu Gln Asp Ala Phe ArgSer Asn Leu Lys Thr Leu Leu Glu 320 325 330 att ctg gac ggg aaa ata tttgaa cta aca gaa ttg cgg gat aat ttg 1780 Ile Leu Asp Gly Lys Ile Phe GluLeu Thr Glu Leu Arg Asp Asn Leu 335 340 345 gcc aag cta cta gaa tgc agctaaagaaagt gaaatttcag tgccaataga 1831 Ala Lys Leu Leu Glu Cys Ser 350tgaagagata ctgtctgtct tcgtaggact gtttgggctc tgtaccaaga ttgcacaaaa 1891ttttttgaat atcattcctc cagaaggagg gtgttttgaa aattggaatt gtatatttca 1951gtataaattt tagaatttag cttatagcta gttgggggaa aaaaagacat gaaaaacttg 2011aaccacaaat tacctccatg tacattggcc atagttacaa tgggagaatt aacaatgtct 2071gggtcccttc tcctttttct gttcaacaca gtgaagatta tctgcttttt aaatttattt 2131acgatatcta cagctgtgtt ttgtgtaaaa acttagtaat ggaagccctg tctttgttgt 2191tatctgaata atttctcagg atattttttt gctgctgaga aagggccatt accaattaat 2251ccttgccagg agttggggag ctatgtctct aattggaatc actataactg ggtgtctgga 2311gttcttccct tttcgtactg agagtgttct cactctagtg actcctctgg tacactccgt 2371gttctccaat cttgtctgtt gtactttact tttccatatt gactccatgt atttatgaga 2431agatattatc tcccatttta ttatacattt tgaagccaac taaacaaagg cagctgagtc 2491cttcagatat ttttcttttt aaatttatag taaatttgac acagaactga aattcagcag 2551tccgtctttg acggtttagt ctagcaatgt taaggatatt tagagaaaat atgcagttac 2611gtttatttat atatttggca agaaattttt tctggatgat caatgctttt caatttatga 2671taaataatgg ttagggggcg ctgtttatta tagataattt taaggtatat agctgttttc 2731aaggaggtcc acttccgtct agcagccaag cagaggactg tatctaaatc gtgatcgtgg 2791cagatgggtc ttcatagaaa ccatgtcttt attcaaactt catagggcaa tattttgaac 2851tgttacctag gcatttcaaa acaggaaata ccgtcaacag actcttctcc aagagcaggt 2911tttactgttg ttttgatgta attttaagac atttagcaaa catgcatttc tttatatgat 2971acatttcttt cacaaaacaa tttaaaagta agccacgtgc tgtctgctct gcccgggtag 3031gaattgcatc agaatacata tatcttgctg tacaatgcct gtgatattga agagggttct 3091tttcatgtat gcttgagtat ctaactctgg agtcaatgaa tgcactgact ttttttttgt 3151tcgtacccca aatgattgaa ttgttaagta caaattaagc agattaactc attttttcac 3211tcataaacag attcttagta ctagttttgt tttatattta tgtgtatgta tgtaaataca 3271tacatattaa tttatattag agtgaaaaat aaattgtttg tttctaacat taaaaaaaaa 3331aaaaaaaa 3339 <210> SEQ ID NO 36 <211> LENGTH: 354 <212> TYPE: PRT <213>ORGANISM: Rattus norvegicus <400> SEQUENCE: 36 Met Gly Glu Gln Pro IlePhe Ser Thr Arg Ala His Val Phe Gln Ile 1 5 10 15 Asp Pro Asn Thr LysLys Asn Trp Val Pro Thr Ser Lys His Ala Val 20 25 30 Thr Val Ser Tyr PheTyr Asp Ser Thr Arg Asn Val Tyr Arg Ile Ile 35 40 45 Ser Leu Asp Gly SerLys Ala Ile Ile Asn Ser Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr LysThr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser 65 70 75 80 Arg Ala Asn ThrVal Tyr Gly Leu Gly Phe Ser Ser Glu His His Leu 85 90 95 Ser Lys Phe AlaGlu Lys Phe Gln Glu Phe Lys Glu Ala Ala Arg Leu 100 105 110 Ala Lys GluLys Ser Gln Glu Lys Met Glu Leu Thr Ser Thr Pro Ser 115 120 125 Gln GluSer Ala Gly Gly Asp Leu Gln Ser Pro Leu Thr Pro Glu Ser 130 135 140 IleAsn Gly Thr Asp Asp Glu Arg Thr Pro Asp Val Thr Gln Asn Ser 145 150 155160 Glu Pro Arg Ala Glu Pro Ala Gln Asn Ala Leu Pro Phe Ser His Ser 165170 175 Ser Ala Ile Ser Lys His Trp Glu Ala Glu Leu Ala Thr Leu Lys Gly180 185 190 Asn Asn Ala Lys Leu Thr Ala Ala Leu Leu Glu Ser Thr Ala AsnVal 195 200 205 Lys Gln Trp Lys Gln Gln Leu Ala Ala Tyr Gln Glu Glu AlaGlu Arg 210 215 220 Leu His Lys Arg Val Thr Glu Leu Glu Cys Val Ser SerGln Ala Asn 225 230 235 240 Ala Val His Ser His Lys Thr Glu Leu Ser GlnThr Val Gln Glu Leu 245 250 255 Glu Glu Thr Leu Lys Val Lys Glu Glu GluIle Glu Arg Leu Lys Gln 260 265 270 Glu Ile Asp Asn Ala Arg Glu Leu GlnGlu Gln Arg Asp Ser Leu Thr 275 280 285 Gln Lys Leu Gln Glu Val Glu IleArg Asn Lys Asp Leu Glu Gly Gln 290 295 300 Leu Ser Glu Leu Glu Gln ArgLeu Glu Lys Ser Gln Ser Glu Gln Asp 305 310 315 320 Ala Phe Arg Ser AsnLeu Lys Thr Leu Leu Glu Ile Leu Asp Gly Lys 325 330 335 Ile Phe Glu LeuThr Glu Leu Arg Asp Asn Leu Ala Lys Leu Leu Glu 340 345 350 Cys Ser<210> SEQ ID NO 37 <211> LENGTH: 3706 <212> TYPE: DNA <213> ORGANISM:Rattus norvegicus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(732)..(1835) <400> SEQUENCE: 37 gcggccgcgt cgactacggc tgcgagaagacgacagaagg gggctccgct gatgctcctc 60 gtgagaacga atcgatcctt cccagccttctctgcctgct ctccacctcc tctctgctcc 120 gagtcttagg agaacgaaca ttcaaaggacagattccaat gtggtgtgct gtgcacatcg 180 cgagcggctg gggtttgcac ttcgagatttcttctttata attttttttt tttaatgtaa 240 gggagacagt ggaattgcta cccgtagaatttttattcaa gtgcacgtcg cgttgggttg 300 cacgctccac ccccagggac ctggtgtggtgaaatttgaa cccaccgcct tagcccaaag 360 gccgagtaac ctggctgctt gagtgtcgtggaagacgtga gcgaaatgat cagcgaactc 420 attttttatc agactcactg aagctggcttttgcgttttt ctacacgtac actaatttta 480 tggaatagtt aaagtgctat attctccgcgcaaccttttc aaattccaaa tgtttgaacg 540 ttttggtgtc agcgcgagtg aaatcattttaccgacaaga actaactgaa ttgtctgcct 600 cgttgagttg cctccggaaa agatctcgggggtggaaaag caactgcaaa ataacagacg 660 gagaaaattc cttggaagtt atttctgtagcataagagca gaaacttcag agcaagtttt 720 cattgggcaa a atg ggg gaa caa cctatc ttc agc act cga gct cat gtc 770 Met Gly Glu Gln Pro Ile Phe Ser ThrArg Ala His Val 1 5 10 ttc cag atc gac cca aac aca aag aag aac tgg gtaccc acc agc aag 818 Phe Gln Ile Asp Pro Asn Thr Lys Lys Asn Trp Val ProThr Ser Lys 15 20 25 cat gca gtt act gtg tct tat ttc tat gac agc aca aggaat gtg tat 866 His Ala Val Thr Val Ser Tyr Phe Tyr Asp Ser Thr Arg AsnVal Tyr 30 35 40 45 agg ata atc agt cta gac ggc tca aag gca ata ata aatagc acc atc 914 Arg Ile Ile Ser Leu Asp Gly Ser Lys Ala Ile Ile Asn SerThr Ile 50 55 60 act cca aac atg aca ttt act aaa aca tct caa aag ttt ggccaa tgg 962 Thr Pro Asn Met Thr Phe Thr Lys Thr Ser Gln Lys Phe Gly GlnTrp 65 70 75 gct gat agc cgg gca aac act gtt tat gga ctg gga ttc tcc tctgag 1010 Ala Asp Ser Arg Ala Asn Thr Val Tyr Gly Leu Gly Phe Ser Ser Glu80 85 90 cat cat ctc tca aaa ttt gca gaa aag ttt cag gaa ttt aaa gaa gct1058 His His Leu Ser Lys Phe Ala Glu Lys Phe Gln Glu Phe Lys Glu Ala 95100 105 gct cgg ctg gca aag gag aag tcg cag gag aag atg gaa ctg acc agt1106 Ala Arg Leu Ala Lys Glu Lys Ser Gln Glu Lys Met Glu Leu Thr Ser 110115 120 125 acc cct tca cag gaa tca gca gga gga gat ctt cag tct cct ttaaca 1154 Thr Pro Ser Gln Glu Ser Ala Gly Gly Asp Leu Gln Ser Pro Leu Thr130 135 140 cca gaa agt atc aat ggg aca gat gat gag aga aca ccc gat gtgaca 1202 Pro Glu Ser Ile Asn Gly Thr Asp Asp Glu Arg Thr Pro Asp Val Thr145 150 155 cag aac tca gag cca agg gct gag cca gct cag aat gca ttg ccattt 1250 Gln Asn Ser Glu Pro Arg Ala Glu Pro Ala Gln Asn Ala Leu Pro Phe160 165 170 tca cat agt gcc ggg gat cga acc cag ggc ctc tct cat gct agttca 1298 Ser His Ser Ala Gly Asp Arg Thr Gln Gly Leu Ser His Ala Ser Ser175 180 185 gcc atc agc aaa cac tgg gag gct gaa cta gcc acg ctc aag gggaac 1346 Ala Ile Ser Lys His Trp Glu Ala Glu Leu Ala Thr Leu Lys Gly Asn190 195 200 205 aat gcc aag ctc acc gca gcg ctg ctg gag tcc act gcc aacgtg aag 1394 Asn Ala Lys Leu Thr Ala Ala Leu Leu Glu Ser Thr Ala Asn ValLys 210 215 220 cag tgg aag caa cag ctg gct gcc tac cag gag gag gca gagcgg ctg 1442 Gln Trp Lys Gln Gln Leu Ala Ala Tyr Gln Glu Glu Ala Glu ArgLeu 225 230 235 cac aag cgg gtc acg gag ctg gaa tgt gtt agt agt caa gcaaac gcg 1490 His Lys Arg Val Thr Glu Leu Glu Cys Val Ser Ser Gln Ala AsnAla 240 245 250 gtg cac agc cac aag aca gag ctg agt cag aca gtg cag gagctg gaa 1538 Val His Ser His Lys Thr Glu Leu Ser Gln Thr Val Gln Glu LeuGlu 255 260 265 gag acc cta aaa gta aag gaa gag gaa ata gaa aga tta aaacaa gaa 1586 Glu Thr Leu Lys Val Lys Glu Glu Glu Ile Glu Arg Leu Lys GlnGlu 270 275 280 285 att gat aac gcc aga gaa ctt caa gaa cag agg gac tctttg act cag 1634 Ile Asp Asn Ala Arg Glu Leu Gln Glu Gln Arg Asp Ser LeuThr Gln 290 295 300 aaa cta cag gaa gtt gag att cga aat aaa gac ctg gagggg cag ctg 1682 Lys Leu Gln Glu Val Glu Ile Arg Asn Lys Asp Leu Glu GlyGln Leu 305 310 315 tcg gag ctg gag cag cgc ctg gag aag agc cag agc gagcag gac gct 1730 Ser Glu Leu Glu Gln Arg Leu Glu Lys Ser Gln Ser Glu GlnAsp Ala 320 325 330 ttc cgc agt aac ctg aag act ctc cta gag att ctg gacggg aaa ata 1778 Phe Arg Ser Asn Leu Lys Thr Leu Leu Glu Ile Leu Asp GlyLys Ile 335 340 345 ttt gaa cta aca gaa ttg cgg gat aat ttg gcc aag ctacta gaa tgc 1826 Phe Glu Leu Thr Glu Leu Arg Asp Asn Leu Ala Lys Leu LeuGlu Cys 350 355 360 365 agc taa aga aagtgaaatt tcagtgccaa tagatgaagagatactgtct 1875 Ser Arg gtcttcgtag gactgtttgg gctctgtacc aagattgcaaaaaatttttt gaatatcatt 1935 cctccagaag gagggtgttt tgaaaattgg aattgtatatttcagtataa attttagaat 1995 ttagcttata gctagttggg ggaaaaaaag acatgaaaaacttgaaccac aaataatgca 2055 atcttttccc ctgatagtag ccaatgggag aattaacaatgtctgggtcc cttctccttt 2115 ttctgttcaa cacagtgaag attatctgct ttttaaatttatttacgata tctacagctg 2175 tgttttgtgt aaaaacttag taatggaagc cctgtctttgttgttatctg aataatttct 2235 caggatattt ttttgctgct gagaaagggc cattaccaattaatccttgc caggagttgg 2295 ggagctatgt ctctaattgg aatcactata actgggtgtctggagttctt cccttttcgt 2355 actgagagtg ttctcactct agtgactact ctggtacactccgtgttctc caatcttgtc 2415 tgttgtactt tacttttcca tattgactcc atgtatttatgagaagatat tatctcccat 2475 tttattatac attttgaagc caactaaaca aaggcagctgagtccttcag atatttttct 2535 ttttaaattt atagtaaatt tgacacagaa ctgaaattcagcagtccgtc tttgacggtt 2595 tagtctagca atgttaagga tatttagaga aaatatgcagttacgtttat ttatatattt 2655 ggcaagaaat tttttctgga tgatcaatgc ttttcaatttatgataaata atggttaggg 2715 gcgctgttta ttatagataa ttttaaggtg tatagctgttttcaaggagg tccactcccg 2775 tctagcagcc aagcagagga ctgtatctaa atcgtgatcgtggcagatgg gtcttcatag 2835 aaaccatgtc tttattcaaa cttcataggg caatattttgaactgttacc taggcatttc 2895 aaaacaggaa ataccgtcaa cagactcttc tccaagagcaggttttactg ttgttttgat 2955 gtaattttaa gacatttagc aaacatgcat ttctttatatgatacatttc tttcacaaaa 3015 caatttaaaa gtaagccacg tgctgtctgc tctgcccgggtaggaattgc atcagaatac 3075 atatatcttg ctgtacaatg cctgtgatat tgaagagggttcttttcatg tatgcttgag 3135 tatctaactc tggagtcaat gaatgcactg acttttttttttgttcgtac cccaaatgat 3195 tgaattgtta agtacaaatt aagcagatta actcattttttcactcataa acagattctt 3255 agtactagtt ttgttttata tttatgtgta tgtatgtaaatacatacata ttaatttata 3315 ttagagtgaa aaataaattg tttgtttcta acattagtttctacagtaag gtgtctctga 3375 aacatgtgtg tcagacactt agccaccatg cattctatgtgctaccccat catgccagtc 3435 acctccatcg acgttagggt attttcctta cctgtctattataaagagaa taacttaggt 3495 acacatgctc agagccgaga tatttctctg ataaatcaggtaataaaatc tatttgatgg 3555 gtagaatttt gaaaacagac atgattttat ctatgagtttctgaatatca aagaacacca 3615 ggttttcatt taaatagagg tctaacacta gggatcagggaatttagtta tgaagagttg 3675 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 3706 <210>SEQ ID NO 38 <211> LENGTH: 366 <212> TYPE: PRT <213> ORGANISM: Rattusnorvegicus <400> SEQUENCE: 38 Met Gly Glu Gln Pro Ile Phe Ser Thr ArgAla His Val Phe Gln Ile 1 5 10 15 Asp Pro Asn Thr Lys Lys Asn Trp ValPro Thr Ser Lys His Ala Val 20 25 30 Thr Val Ser Tyr Phe Tyr Asp Ser ThrArg Asn Val Tyr Arg Ile Ile 35 40 45 Ser Leu Asp Gly Ser Lys Ala Ile IleAsn Ser Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr Lys Thr Ser Gln LysPhe Gly Gln Trp Ala Asp Ser 65 70 75 80 Arg Ala Asn Thr Val Tyr Gly LeuGly Phe Ser Ser Glu His His Leu 85 90 95 Ser Lys Phe Ala Glu Lys Phe GlnGlu Phe Lys Glu Ala Ala Arg Leu 100 105 110 Ala Lys Glu Lys Ser Gln GluLys Met Glu Leu Thr Ser Thr Pro Ser 115 120 125 Gln Glu Ser Ala Gly GlyAsp Leu Gln Ser Pro Leu Thr Pro Glu Ser 130 135 140 Ile Asn Gly Thr AspAsp Glu Arg Thr Pro Asp Val Thr Gln Asn Ser 145 150 155 160 Glu Pro ArgAla Glu Pro Ala Gln Asn Ala Leu Pro Phe Ser His Ser 165 170 175 Ala GlyAsp Arg Thr Gln Gly Leu Ser His Ala Ser Ser Ala Ile Ser 180 185 190 LysHis Trp Glu Ala Glu Leu Ala Thr Leu Lys Gly Asn Asn Ala Lys 195 200 205Leu Thr Ala Ala Leu Leu Glu Ser Thr Ala Asn Val Lys Gln Trp Lys 210 215220 Gln Gln Leu Ala Ala Tyr Gln Glu Glu Ala Glu Arg Leu His Lys Arg 225230 235 240 Val Thr Glu Leu Glu Cys Val Ser Ser Gln Ala Asn Ala Val HisSer 245 250 255 His Lys Thr Glu Leu Ser Gln Thr Val Gln Glu Leu Glu GluThr Leu 260 265 270 Lys Val Lys Glu Glu Glu Ile Glu Arg Leu Lys Gln GluIle Asp Asn 275 280 285 Ala Arg Glu Leu Gln Glu Gln Arg Asp Ser Leu ThrGln Lys Leu Gln 290 295 300 Glu Val Glu Ile Arg Asn Lys Asp Leu Glu GlyGln Leu Ser Glu Leu 305 310 315 320 Glu Gln Arg Leu Glu Lys Ser Gln SerGlu Gln Asp Ala Phe Arg Ser 325 330 335 Asn Leu Lys Thr Leu Leu Glu IleLeu Asp Gly Lys Ile Phe Glu Leu 340 345 350 Thr Glu Leu Arg Asp Asn LeuAla Lys Leu Leu Glu Cys Ser 355 360 365 <210> SEQ ID NO 39 <211> LENGTH:7424 <212> TYPE: DNA <213> ORGANISM: Rattus norvegicus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (267)..(5486) <400> SEQUENCE: 39ctctagaact agtggatccc ccgggctgca ggattctgcg gccgcgctaa accgtgccgc 60cgtcgccgcc gccgctgcgc ctgcggagcc cccggagccg ctgtcccccg cgctggcccc 120ggccccggcc ccatacggcc ccctcccgca gtagcgcggt cggcgggact ctggcggggg 180gtcagggggg gccagggcgc cgcgcggagt ccccgtgcgc tcctctctcc gccgggaaca 240gtccgggccc cggcgctagc accggg atg gac ggc ccc ggg gcc agc gcc gtg 293 MetAsp Gly Pro Gly Ala Ser Ala Val 1 5 gtc gtg cgc gtc ggc atc ccg gac ctgcaa caa acg aag tgc ctg cgt 341 Val Val Arg Val Gly Ile Pro Asp Leu GlnGln Thr Lys Cys Leu Arg 10 15 20 25 ctg gat cca acc gcg ccc gtg tgg gccgcc aag cag cgt gtg ctc tgc 389 Leu Asp Pro Thr Ala Pro Val Trp Ala AlaLys Gln Arg Val Leu Cys 30 35 40 gcc ctc aac cac agc ctt cag gac gcg ctcaac tac ggg cta ttc cag 437 Ala Leu Asn His Ser Leu Gln Asp Ala Leu AsnTyr Gly Leu Phe Gln 45 50 55 cct ccc tcc cgg ggt cgc gcc ggc aag ttc ctggat gaa gag cgg ctc 485 Pro Pro Ser Arg Gly Arg Ala Gly Lys Phe Leu AspGlu Glu Arg Leu 60 65 70 tta cag gac tac ccg cct aac ctg gac acg ccc ctgccc tat ctg gag 533 Leu Gln Asp Tyr Pro Pro Asn Leu Asp Thr Pro Leu ProTyr Leu Glu 75 80 85 ttt cga tac aag cgg aga gtt tat gcc cag aac ctc atagat gac aag 581 Phe Arg Tyr Lys Arg Arg Val Tyr Ala Gln Asn Leu Ile AspAsp Lys 90 95 100 105 cag ttt gca aag ctg cac aca aag gca aac ctg aagaag ttc atg gac 629 Gln Phe Ala Lys Leu His Thr Lys Ala Asn Leu Lys LysPhe Met Asp 110 115 120 tat gtc cag cta cac agc aca gac aag gtg gcc cgcctg ctg gac aag 677 Tyr Val Gln Leu His Ser Thr Asp Lys Val Ala Arg LeuLeu Asp Lys 125 130 135 ggg ctg gac ccc aat ttc cat gac cct gac tca ggagag tgc cct ctg 725 Gly Leu Asp Pro Asn Phe His Asp Pro Asp Ser Gly GluCys Pro Leu 140 145 150 agc ctt gca gca cag ttg gac aac gcc act gac ctcctg aag gtt ctt 773 Ser Leu Ala Ala Gln Leu Asp Asn Ala Thr Asp Leu LeuLys Val Leu 155 160 165 cgc aat ggc ggt gct cat ctg gac ttc cga acc cgagat ggg cta acc 821 Arg Asn Gly Gly Ala His Leu Asp Phe Arg Thr Arg AspGly Leu Thr 170 175 180 185 gct gtc cac tgc gcc acc cga cag cgg aat gcggga gca ttg acg acc 869 Ala Val His Cys Ala Thr Arg Gln Arg Asn Ala GlyAla Leu Thr Thr 190 195 200 ctg ctg gac ctg ggg gct tca cct gac tac aaggac agc cgc ggc ctg 917 Leu Leu Asp Leu Gly Ala Ser Pro Asp Tyr Lys AspSer Arg Gly Leu 205 210 215 acg ccc ctg tac cat agt gcc cta ggg ggc ggggat gcc ctc tgc tgt 965 Thr Pro Leu Tyr His Ser Ala Leu Gly Gly Gly AspAla Leu Cys Cys 220 225 230 gag ctg ctt ctc cat gat cac gca cag ttg gggacc act gac gag aat 1013 Glu Leu Leu Leu His Asp His Ala Gln Leu Gly ThrThr Asp Glu Asn 235 240 245 ggc tgg cag gag atc cat cag gcc tgt cgc tttggg cat gta cag cac 1061 Gly Trp Gln Glu Ile His Gln Ala Cys Arg Phe GlyHis Val Gln His 250 255 260 265 ttg gag cac ctg ctg ttc tat ggg gcc aacatg ggt gcc cag aac gcc 1109 Leu Glu His Leu Leu Phe Tyr Gly Ala Asn MetGly Ala Gln Asn Ala 270 275 280 tcg gga aac aca gcc ttg cac atc tgt gccctc tat aac cag gag agc 1157 Ser Gly Asn Thr Ala Leu His Ile Cys Ala LeuTyr Asn Gln Glu Ser 285 290 295 tgt gcc cgc gtc ctg ctt ttc cgt ggt gccaac aag gac gtc cgc aat 1205 Cys Ala Arg Val Leu Leu Phe Arg Gly Ala AsnLys Asp Val Arg Asn 300 305 310 tac aac agc cag aca gcc ttc cag gtg gccatt att gca ggg aac ttt 1253 Tyr Asn Ser Gln Thr Ala Phe Gln Val Ala IleIle Ala Gly Asn Phe 315 320 325 gag ctt gcc gag gta atc aag acc cac aaagac tcg gat gtc gta cca 1301 Glu Leu Ala Glu Val Ile Lys Thr His Lys AspSer Asp Val Val Pro 330 335 340 345 ttc agg gaa acc ccc agc tat gca aagcga cga cgt ctg gct ggc ccg 1349 Phe Arg Glu Thr Pro Ser Tyr Ala Lys ArgArg Arg Leu Ala Gly Pro 350 355 360 agt ggc ttg gca tcc cct cgg ccc ttacag cgc tca gcc agt gat atc 1397 Ser Gly Leu Ala Ser Pro Arg Pro Leu GlnArg Ser Ala Ser Asp Ile 365 370 375 aac ctg aag ggt gac cag ccc gca gcttct ccc ggg ccc act ctc cga 1445 Asn Leu Lys Gly Asp Gln Pro Ala Ala SerPro Gly Pro Thr Leu Arg 380 385 390 agc ctc cct cac caa ctg ctg ctc cagagg ctt cag gag gag aaa gac 1493 Ser Leu Pro His Gln Leu Leu Leu Gln ArgLeu Gln Glu Glu Lys Asp 395 400 405 cgg gac agg gat ggt gag cag gag aacgac atc agc ggt ccc tca gca 1541 Arg Asp Arg Asp Gly Glu Gln Glu Asn AspIle Ser Gly Pro Ser Ala 410 415 420 425 ggc agg ggc ggc cac agc aag atcagc ccc agc ggg ccc ggc gga tcc 1589 Gly Arg Gly Gly His Ser Lys Ile SerPro Ser Gly Pro Gly Gly Ser 430 435 440 ggc ccc gcg ccc ggc ccc ggc ccggcg tct ccc gcg ccc ccc gcg ccg 1637 Gly Pro Ala Pro Gly Pro Gly Pro AlaSer Pro Ala Pro Pro Ala Pro 445 450 455 ccg ccc cgg ggc ccg aag cgg aaactt tac agt gcc gtc ccc ggc cgc 1685 Pro Pro Arg Gly Pro Lys Arg Lys LeuTyr Ser Ala Val Pro Gly Arg 460 465 470 aag ttc atc gct gtg aag gcg cacagc ccg cag ggc gag ggc gag atc 1733 Lys Phe Ile Ala Val Lys Ala His SerPro Gln Gly Glu Gly Glu Ile 475 480 485 ccg ctg cac cgc ggc gag gcc gtgaag gtg ctc agc att ggg gag ggc 1781 Pro Leu His Arg Gly Glu Ala Val LysVal Leu Ser Ile Gly Glu Gly 490 495 500 505 ggt ttc tgg gag gga acc gtgaag ggc cgt aca ggc tgg ttc cca gct 1829 Gly Phe Trp Glu Gly Thr Val LysGly Arg Thr Gly Trp Phe Pro Ala 510 515 520 gac tgt gtg gag gaa gtg cagatg cga cag tat gac aca cgg cat gaa 1877 Asp Cys Val Glu Glu Val Gln MetArg Gln Tyr Asp Thr Arg His Glu 525 530 535 act cga gag gac cgg acg aagcgt ctt ttc cgc cac tac act gtg ggt 1925 Thr Arg Glu Asp Arg Thr Lys ArgLeu Phe Arg His Tyr Thr Val Gly 540 545 550 tcc tat gac agc ctc act tcacac agt gat tat gtc att gat gat aag 1973 Ser Tyr Asp Ser Leu Thr Ser HisSer Asp Tyr Val Ile Asp Asp Lys 555 560 565 gtg gct atc ctg caa aaa cgggac cat gag ggt ttt ggc ttt gtt ctc 2021 Val Ala Ile Leu Gln Lys Arg AspHis Glu Gly Phe Gly Phe Val Leu 570 575 580 585 cgg gga gcc aaa gca gagacc ccc att gag gag ttt aca ccc aca cct 2069 Arg Gly Ala Lys Ala Glu ThrPro Ile Glu Glu Phe Thr Pro Thr Pro 590 595 600 gcc ttc cct gcg ctc cagtac ctt gag tct gta gat gtg gaa ggt gtg 2117 Ala Phe Pro Ala Leu Gln TyrLeu Glu Ser Val Asp Val Glu Gly Val 605 610 615 gcc tgg aag gct ggg cttcgc act ggg gac ttc ctc att gag gta aac 2165 Ala Trp Lys Ala Gly Leu ArgThr Gly Asp Phe Leu Ile Glu Val Asn 620 625 630 gga gtg aac gtc gtg aaggtt gga cac aag caa gtg gtg ggt ctc atc 2213 Gly Val Asn Val Val Lys ValGly His Lys Gln Val Val Gly Leu Ile 635 640 645 cgt cag ggt ggc aac cgtctg gtc atg aag gtt gtg tct gtt acc agg 2261 Arg Gln Gly Gly Asn Arg LeuVal Met Lys Val Val Ser Val Thr Arg 650 655 660 665 aag cca gag gag gatagt gct cgg cgc aga gcc cca cca cct ccc aag 2309 Lys Pro Glu Glu Asp SerAla Arg Arg Arg Ala Pro Pro Pro Pro Lys 670 675 680 agg gcc ccc agc accacg ctg acc ctg cgg tcc aag tcc atg acg gct 2357 Arg Ala Pro Ser Thr ThrLeu Thr Leu Arg Ser Lys Ser Met Thr Ala 685 690 695 gag ctc gag gaa ctcgct tcc att cgg aga agg aaa ggg gag aag ttg 2405 Glu Leu Glu Glu Leu AlaSer Ile Arg Arg Arg Lys Gly Glu Lys Leu 700 705 710 gat gag atc ctg gcggtt gct gcg gaa cca acg ctg agg cca gac att 2453 Asp Glu Ile Leu Ala ValAla Ala Glu Pro Thr Leu Arg Pro Asp Ile 715 720 725 gca gac gct gat tccagg gca gcc act gtc aag cag cgg ccc acc agc 2501 Ala Asp Ala Asp Ser ArgAla Ala Thr Val Lys Gln Arg Pro Thr Ser 730 735 740 745 cgg agg att acccct gcc gag atc agc tca ttg ttt gag cga cag ggc 2549 Arg Arg Ile Thr ProAla Glu Ile Ser Ser Leu Phe Glu Arg Gln Gly 750 755 760 ctc ccg ggc ccagag aag ctg ccg ggc tct ctg cgg aag ggg att cca 2597 Leu Pro Gly Pro GluLys Leu Pro Gly Ser Leu Arg Lys Gly Ile Pro 765 770 775 cgg acc aaa tctgta ggg gag gat gag aag ctg gca tcc cta ctg gaa 2645 Arg Thr Lys Ser ValGly Glu Asp Glu Lys Leu Ala Ser Leu Leu Glu 780 785 790 ggg cgt ttc ccacgc agc aca tca atg caa gac aca gtg cgt gaa ggc 2693 Gly Arg Phe Pro ArgSer Thr Ser Met Gln Asp Thr Val Arg Glu Gly 795 800 805 cga ggc att ccgccc cca ccg cag acc gcc ccg cca ccc cca ccc gcg 2741 Arg Gly Ile Pro ProPro Pro Gln Thr Ala Pro Pro Pro Pro Pro Ala 810 815 820 825 ccc tac tacttc gac tcc ggg cca ccc ccc acc ttc tca cca ccg cca 2789 Pro Tyr Tyr PheAsp Ser Gly Pro Pro Pro Thr Phe Ser Pro Pro Pro 830 835 840 cca cca ccgggc cgg gcc tat gac act gtg cgc tcc agc ttc aag cca 2837 Pro Pro Pro GlyArg Ala Tyr Asp Thr Val Arg Ser Ser Phe Lys Pro 845 850 855 ggc ctg gaggct cgt ctg ggt gca ggg gca gct ggc ctg tat gat tct 2885 Gly Leu Glu AlaArg Leu Gly Ala Gly Ala Ala Gly Leu Tyr Asp Ser 860 865 870 ggc aca cctctg ggc ccg ctg ccc tac cct gag cgc cag aag cgt gca 2933 Gly Thr Pro LeuGly Pro Leu Pro Tyr Pro Glu Arg Gln Lys Arg Ala 875 880 885 cgc tcc atgatc ata ttg cag gac tct gcg cca gaa gtg ggc gat gta 2981 Arg Ser Met IleIle Leu Gln Asp Ser Ala Pro Glu Val Gly Asp Val 890 895 900 905 ccc cggcct gcg cct gca gcc aca ccg cct gag cgc ccc aag cgc cgg 3029 Pro Arg ProAla Pro Ala Ala Thr Pro Pro Glu Arg Pro Lys Arg Arg 910 915 920 cct cggccg tca ggc cct gat agt ccc tat gcc aac ctg ggc gcc ttc 3077 Pro Arg ProSer Gly Pro Asp Ser Pro Tyr Ala Asn Leu Gly Ala Phe 925 930 935 agt gccagc ctc ttt gct ccg tcg aaa ccg cag cgc cgc aag agt ccg 3125 Ser Ala SerLeu Phe Ala Pro Ser Lys Pro Gln Arg Arg Lys Ser Pro 940 945 950 ctg gtgaag cag ctt cag gtg gag gac gct cag gag cgc gcg gcg ttg 3173 Leu Val LysGln Leu Gln Val Glu Asp Ala Gln Glu Arg Ala Ala Leu 955 960 965 gcc gtgggt agc ccg gga cca gtg ggt gga agc ttt gca cga gaa ccc 3221 Ala Val GlySer Pro Gly Pro Val Gly Gly Ser Phe Ala Arg Glu Pro 970 975 980 985 tcccca acg cac cgc ggg ccc cga ccg ggc ggc ctt gac tac agc tct 3269 Ser ProThr His Arg Gly Pro Arg Pro Gly Gly Leu Asp Tyr Ser Ser 990 995 1000 ggagaa ggc ctg ggg ctc acc ttt ggc ggc cct agc cct ggc cca 3314 Gly Glu GlyLeu Gly Leu Thr Phe Gly Gly Pro Ser Pro Gly Pro 1005 1010 1015 gtc aaggag cgg cgc ctg gag gag cga cgc cgt tcc act gtg ttc 3359 Val Lys Glu ArgArg Leu Glu Glu Arg Arg Arg Ser Thr Val Phe 1020 1025 1030 ctg tct gtgggt gcc atc gag ggc aac cct ccc agc gcg gat ctg 3404 Leu Ser Val Gly AlaIle Glu Gly Asn Pro Pro Ser Ala Asp Leu 1035 1040 1045 cca tcc cta caaccc tcc cgc tcc att gat gag cgc ctc ctg ggg 3449 Pro Ser Leu Gln Pro SerArg Ser Ile Asp Glu Arg Leu Leu Gly 1050 1055 1060 aca ggc gcc acc actggc cga gat ttg ctg ctc ccc tcc cct gtc 3494 Thr Gly Ala Thr Thr Gly ArgAsp Leu Leu Leu Pro Ser Pro Val 1065 1070 1075 tct gct ctg aag cca ttggtc ggt ggt ccc aac ctt ggg ccc tca 3539 Ser Ala Leu Lys Pro Leu Val GlyGly Pro Asn Leu Gly Pro Ser 1080 1085 1090 agc tcc acc ttc atc cat cctctt act ggc aaa ccc ttg gat cct 3584 Ser Ser Thr Phe Ile His Pro Leu ThrGly Lys Pro Leu Asp Pro 1095 1100 1105 agc tca ccc cta gct ctt gct ctggct gcc cga gag cgg gct ctg 3629 Ser Ser Pro Leu Ala Leu Ala Leu Ala AlaArg Glu Arg Ala Leu 1110 1115 1120 gcc tcg caa aca cct tcc cgg tcc cccaca ccc gtg cac agt cct 3674 Ala Ser Gln Thr Pro Ser Arg Ser Pro Thr ProVal His Ser Pro 1125 1130 1135 gat gct gac cgc cct gga ccc ctc ttt gtggat gtg caa acc cga 3719 Asp Ala Asp Arg Pro Gly Pro Leu Phe Val Asp ValGln Thr Arg 1140 1145 1150 gac tcc gag aga gga ccc ttg gcc tcc cca gccttc tcc cct cgg 3764 Asp Ser Glu Arg Gly Pro Leu Ala Ser Pro Ala Phe SerPro Arg 1155 1160 1165 agt cca gcc tgg att cca gtg cct gct cgc aga gaggca gag aag 3809 Ser Pro Ala Trp Ile Pro Val Pro Ala Arg Arg Glu Ala GluLys 1170 1175 1180 ccc act cgg gaa gag cgg aag tca cca gag gac aag aaatcc atg 3854 Pro Thr Arg Glu Glu Arg Lys Ser Pro Glu Asp Lys Lys Ser Met1185 1190 1195 atc ctc agc gtc ttg gac acg tcc ttg caa cgg cca gct ggcctc 3899 Ile Leu Ser Val Leu Asp Thr Ser Leu Gln Arg Pro Ala Gly Leu1200 1205 1210 att gtt gtg cat gcc acc agc aat gga cag gag ccc aac aggctg 3944 Ile Val Val His Ala Thr Ser Asn Gly Gln Glu Pro Asn Arg Leu1215 1220 1225 ggg gct gaa gag gag cgc ccg ggt act ccg gag ctg gcc ccaacc 3989 Gly Ala Glu Glu Glu Arg Pro Gly Thr Pro Glu Leu Ala Pro Thr1230 1235 1240 ccc atg cag gca gca gct gtg gca gag ccc atg cca agc ccacga 4034 Pro Met Gln Ala Ala Ala Val Ala Glu Pro Met Pro Ser Pro Arg1245 1250 1255 gcc caa ccc cct ggc aac atc cca gca gat ccc ggg cca agccaa 4079 Ala Gln Pro Pro Gly Asn Ile Pro Ala Asp Pro Gly Pro Ser Gln1260 1265 1270 ggc aac tca gag gag gag cca aag ctg gta ttc gct gtg aacctg 4124 Gly Asn Ser Glu Glu Glu Pro Lys Leu Val Phe Ala Val Asn Leu1275 1280 1285 cca cct gct caa ctg tcc tcc aac gat gag gag acc aga gaggag 4169 Pro Pro Ala Gln Leu Ser Ser Asn Asp Glu Glu Thr Arg Glu Glu1290 1295 1300 ctg gcc cgc att ggg cta gtg cca ccc cct gaa gag ttt gccaat 4214 Leu Ala Arg Ile Gly Leu Val Pro Pro Pro Glu Glu Phe Ala Asn1305 1310 1315 ggg atc ctg ctg gcc acc cca ccc cca gga ccg ggc ccc ttgccc 4259 Gly Ile Leu Leu Ala Thr Pro Pro Pro Gly Pro Gly Pro Leu Pro1320 1325 1330 acc acg gta ccc agc ccg gcc tca ggg aag ccc agc agc gagctg 4304 Thr Thr Val Pro Ser Pro Ala Ser Gly Lys Pro Ser Ser Glu Leu1335 1340 1345 ccc cct gcc ccg gag tct gca gct gac tct gga gta gag gaggcc 4349 Pro Pro Ala Pro Glu Ser Ala Ala Asp Ser Gly Val Glu Glu Ala1350 1355 1360 gac act cga agc tcc agt gac ccc cac ctg gag acc aca agcacc 4394 Asp Thr Arg Ser Ser Ser Asp Pro His Leu Glu Thr Thr Ser Thr1365 1370 1375 att tcc aca gtg tcc agc atg tcc acc ctg agc tcg gag agtgga 4439 Ile Ser Thr Val Ser Ser Met Ser Thr Leu Ser Ser Glu Ser Gly1380 1385 1390 gaa ctc act gac acc cac acc tcc ttt gcc gat gga cac actttt 4484 Glu Leu Thr Asp Thr His Thr Ser Phe Ala Asp Gly His Thr Phe1395 1400 1405 cta ctc gag aag cca cca gtg cct ccc aag ccc aaa ctc aagtcc 4529 Leu Leu Glu Lys Pro Pro Val Pro Pro Lys Pro Lys Leu Lys Ser1410 1415 1420 ccg ctg ggg aag ggg ccg gtg acc ttc agg ggc ccg ctg ctgaag 4574 Pro Leu Gly Lys Gly Pro Val Thr Phe Arg Gly Pro Leu Leu Lys1425 1430 1435 caa tcc tcg gac agt gag ctc atg gcc cag cag cac cat gccacc 4619 Gln Ser Ser Asp Ser Glu Leu Met Ala Gln Gln His His Ala Thr1440 1445 1450 tct act ggg ttg act tct gct gct ggg cct gcc cgc cct cgctac 4664 Ser Thr Gly Leu Thr Ser Ala Ala Gly Pro Ala Arg Pro Arg Tyr1455 1460 1465 ctc ttc cag aga agg tcc aag ctg tgg ggg gac ccc gtg gagagt 4709 Leu Phe Gln Arg Arg Ser Lys Leu Trp Gly Asp Pro Val Glu Ser1470 1475 1480 cgg ggg ctc cct ggg cct gag gat gac aaa cca act gtg atcagt 4754 Arg Gly Leu Pro Gly Pro Glu Asp Asp Lys Pro Thr Val Ile Ser1485 1490 1495 gag ctc agc tcc cgt ctg cag cag ctg aat aaa gac act cgctcc 4799 Glu Leu Ser Ser Arg Leu Gln Gln Leu Asn Lys Asp Thr Arg Ser1500 1505 1510 ttg ggg gag gaa cca gtt ggt ggc ctg ggt agc ctg ctg gaccct 4844 Leu Gly Glu Glu Pro Val Gly Gly Leu Gly Ser Leu Leu Asp Pro1515 1520 1525 gct aag aag tcg ccc att gca gca gct cgc tgc gcg gtg gtcccg 4889 Ala Lys Lys Ser Pro Ile Ala Ala Ala Arg Cys Ala Val Val Pro1530 1535 1540 agt gcc ggc tgg ctc ttc agc agc ctc ggt gag ctg agc accatc 4934 Ser Ala Gly Trp Leu Phe Ser Ser Leu Gly Glu Leu Ser Thr Ile1545 1550 1555 tca gcg cag cgc agc ccc ggg ggc ccg ggc gga ggg gcc tcctac 4979 Ser Ala Gln Arg Ser Pro Gly Gly Pro Gly Gly Gly Ala Ser Tyr1560 1565 1570 tcg gtg cgg ccc agc ggc cgg tac ccc gtg gcg aga cga gccccg 5024 Ser Val Arg Pro Ser Gly Arg Tyr Pro Val Ala Arg Arg Ala Pro1575 1580 1585 agc cca gtg aaa ccc gca tcg ctg gag cgg gtg gag ggg ctgggg 5069 Ser Pro Val Lys Pro Ala Ser Leu Glu Arg Val Glu Gly Leu Gly1590 1595 1600 gcg ggc gtg gga ggc gcg ggg cgg ccc ttc ggc ctc acg cctccc 5114 Ala Gly Val Gly Gly Ala Gly Arg Pro Phe Gly Leu Thr Pro Pro1605 1610 1615 acc atc ctc aag tcg tcc agc ctc tcc atc ccg cac gaa cccaag 5159 Thr Ile Leu Lys Ser Ser Ser Leu Ser Ile Pro His Glu Pro Lys1620 1625 1630 gaa gtg cgc ttc gtg gtg cga agt gcg agt gcg cgc agc cgctcc 5204 Glu Val Arg Phe Val Val Arg Ser Ala Ser Ala Arg Ser Arg Ser1635 1640 1645 ccc tca cca tct ccg ctg ccc tcg cct tct cct ggc tct ggcccc 5249 Pro Ser Pro Ser Pro Leu Pro Ser Pro Ser Pro Gly Ser Gly Pro1650 1655 1660 agt gcc ggc ccg cgt cgg cca ttt caa cag aag ccc ctg cagctt 5294 Ser Ala Gly Pro Arg Arg Pro Phe Gln Gln Lys Pro Leu Gln Leu1665 1670 1675 tgg agc aag ttc gat gtg ggc gac tgg ctg gag agc atc cactta 5339 Trp Ser Lys Phe Asp Val Gly Asp Trp Leu Glu Ser Ile His Leu1680 1685 1690 ggc gag cac cga gac cgc ttc gag gac cat gag atc gaa ggcgca 5384 Gly Glu His Arg Asp Arg Phe Glu Asp His Glu Ile Glu Gly Ala1695 1700 1705 cac ctg cct gcg ctc acc aag gaa gac ttc gtg gag ctg ggagtc 5429 His Leu Pro Ala Leu Thr Lys Glu Asp Phe Val Glu Leu Gly Val1710 1715 1720 aca cgc gtt ggc cac cgc atg aac atc gag cgt gcg ctc aggcag 5474 Thr Arg Val Gly His Arg Met Asn Ile Glu Arg Ala Leu Arg Gln1725 1730 1735 ctg gat ggc agc tgacgcccct ctccctctcc tgttcctgctgcgccctgcc 5526 Leu Asp Gly Ser 1740 ggcagggccc ccacccctac tccaggccgcaggctcggct cgccccctac cacggcgccc 5586 gggccaggaa tgttgcatga atcgtcctgtttgctgttgc ttggagactt gccctgtaca 5646 ttgcttagtg ccctcccctg ccgctgaaccccacccagca cacagtaagg gcgcggacca 5706 ggggggctgg gtggaagggg gttggggcagggtgctctgg cctgaccacc tcctccacag 5766 ctcctggtgg ccattcttcc agagggggaacctagtccag catgcgaggt caggacacgc 5826 cttggtgact cggggggagg ggggagacattggggttctc gataggggcc aaggagcccc 5886 ctgttttaca tattttaatc cactctatatttggaaagag aaaaggaaca aatatctctg 5946 tccgtaacag ttcccgccct cttcccctcaagtcctctcg ctggtcccgc cacagctacc 6006 cagtcttcca tctccggccc ctcactgccaccccatatag ggcaggggac actccagctg 6066 gcctggggtt agccagggtc ctggcagcccaccctgggga ccccggctca gcccccttcc 6126 ctcgctgagc tatagtatgc cccacccaccctttaggtgc tgctcagggg gacgggtggc 6186 aggcattgcc tgctgggcac tagcagggccaggtggcctg ggagattatt gccctggggc 6246 tgggccccgg taacccaacc ccagccatcatcttcacagg gtctctccca aaggaggggt 6306 ctaacctttc cccacttctt gggcaactacagcagagaag cctccctgcc tcgcgcccca 6366 aagactcccc aattcctgcc ctgtgtgtgtgcaccacatg tgtgtgtgca cgcctgcgtg 6426 cttgtgaaaa ttgggtgtgg ctgagcgcatgggtgccctg tatgtgcttg attgtggagt 6486 ggtccccagg ggctgttctg gatgggtgggaggttgagga agcttgcaca ggggtgcatg 6546 catgggtgtg tgcctgtgaa agggccctgtcttctccaaa gaaaggctgt cctgctcttg 6606 ggtcctgctg ttttctcagc ctgttctccctgaacctcac ccagcttaag caggggttct 6666 tggtgaatcc tttcagcttt gggaggcctcaagggctccc gtgcaggcag cacccctttg 6726 ggcttctaag ggaattgtgg ggaccactaaaatcaggcca caacagccct tggagagagg 6786 caaagactcc tgagggtacc ctggccccccttactgtgac tcctcacaat tcagcaatga 6846 cctgtggggc ggggggcctt ggggcatttttaacataggg tttggagtct ggactaagct 6906 ccatccacgt cactcacaag tttctgtttctatttctagc tttttttaat aaaatatata 6966 tatatatata tataaaagac agaaaacaggtgttttcatg gcccaggggc ttggcacgcc 7026 ggtctgtgcc cacccgcccc gccccaccctggcccaccgg ccccattcct tagacacaga 7086 gtcacgccca ctaaccctct taccaacagagcaggtcaca cacacagcag cggtcactgt 7146 aacagactgc cacatacaca gtctcacatttacctgtggg tttttggttc tgttcagttt 7206 gggtttttaa ctttacaggg tcagttccgcttcatccccc ttttgtatgg agttccatct 7266 cggggctttc aaccccctgc tccagtcctgaggcctcctg accctgacgt tgtgatacac 7326 cccacagaga tctatgtttc ttatattattattattaata ataattatta taatattatg 7386 taataaattt ataagaaatg aaaaaaaaaaaaaaaaaa 7424 <210> SEQ ID NO 40 <211> LENGTH: 1740 <212> TYPE: PRT<213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 40 Met Asp Gly Pro GlyAla Ser Ala Val Val Val Arg Val Gly Ile Pro 1 5 10 15 Asp Leu Gln GlnThr Lys Cys Leu Arg Leu Asp Pro Thr Ala Pro Val 20 25 30 Trp Ala Ala LysGln Arg Val Leu Cys Ala Leu Asn His Ser Leu Gln 35 40 45 Asp Ala Leu AsnTyr Gly Leu Phe Gln Pro Pro Ser Arg Gly Arg Ala 50 55 60 Gly Lys Phe LeuAsp Glu Glu Arg Leu Leu Gln Asp Tyr Pro Pro Asn 65 70 75 80 Leu Asp ThrPro Leu Pro Tyr Leu Glu Phe Arg Tyr Lys Arg Arg Val 85 90 95 Tyr Ala GlnAsn Leu Ile Asp Asp Lys Gln Phe Ala Lys Leu His Thr 100 105 110 Lys AlaAsn Leu Lys Lys Phe Met Asp Tyr Val Gln Leu His Ser Thr 115 120 125 AspLys Val Ala Arg Leu Leu Asp Lys Gly Leu Asp Pro Asn Phe His 130 135 140Asp Pro Asp Ser Gly Glu Cys Pro Leu Ser Leu Ala Ala Gln Leu Asp 145 150155 160 Asn Ala Thr Asp Leu Leu Lys Val Leu Arg Asn Gly Gly Ala His Leu165 170 175 Asp Phe Arg Thr Arg Asp Gly Leu Thr Ala Val His Cys Ala ThrArg 180 185 190 Gln Arg Asn Ala Gly Ala Leu Thr Thr Leu Leu Asp Leu GlyAla Ser 195 200 205 Pro Asp Tyr Lys Asp Ser Arg Gly Leu Thr Pro Leu TyrHis Ser Ala 210 215 220 Leu Gly Gly Gly Asp Ala Leu Cys Cys Glu Leu LeuLeu His Asp His 225 230 235 240 Ala Gln Leu Gly Thr Thr Asp Glu Asn GlyTrp Gln Glu Ile His Gln 245 250 255 Ala Cys Arg Phe Gly His Val Gln HisLeu Glu His Leu Leu Phe Tyr 260 265 270 Gly Ala Asn Met Gly Ala Gln AsnAla Ser Gly Asn Thr Ala Leu His 275 280 285 Ile Cys Ala Leu Tyr Asn GlnGlu Ser Cys Ala Arg Val Leu Leu Phe 290 295 300 Arg Gly Ala Asn Lys AspVal Arg Asn Tyr Asn Ser Gln Thr Ala Phe 305 310 315 320 Gln Val Ala IleIle Ala Gly Asn Phe Glu Leu Ala Glu Val Ile Lys 325 330 335 Thr His LysAsp Ser Asp Val Val Pro Phe Arg Glu Thr Pro Ser Tyr 340 345 350 Ala LysArg Arg Arg Leu Ala Gly Pro Ser Gly Leu Ala Ser Pro Arg 355 360 365 ProLeu Gln Arg Ser Ala Ser Asp Ile Asn Leu Lys Gly Asp Gln Pro 370 375 380Ala Ala Ser Pro Gly Pro Thr Leu Arg Ser Leu Pro His Gln Leu Leu 385 390395 400 Leu Gln Arg Leu Gln Glu Glu Lys Asp Arg Asp Arg Asp Gly Glu Gln405 410 415 Glu Asn Asp Ile Ser Gly Pro Ser Ala Gly Arg Gly Gly His SerLys 420 425 430 Ile Ser Pro Ser Gly Pro Gly Gly Ser Gly Pro Ala Pro GlyPro Gly 435 440 445 Pro Ala Ser Pro Ala Pro Pro Ala Pro Pro Pro Arg GlyPro Lys Arg 450 455 460 Lys Leu Tyr Ser Ala Val Pro Gly Arg Lys Phe IleAla Val Lys Ala 465 470 475 480 His Ser Pro Gln Gly Glu Gly Glu Ile ProLeu His Arg Gly Glu Ala 485 490 495 Val Lys Val Leu Ser Ile Gly Glu GlyGly Phe Trp Glu Gly Thr Val 500 505 510 Lys Gly Arg Thr Gly Trp Phe ProAla Asp Cys Val Glu Glu Val Gln 515 520 525 Met Arg Gln Tyr Asp Thr ArgHis Glu Thr Arg Glu Asp Arg Thr Lys 530 535 540 Arg Leu Phe Arg His TyrThr Val Gly Ser Tyr Asp Ser Leu Thr Ser 545 550 555 560 His Ser Asp TyrVal Ile Asp Asp Lys Val Ala Ile Leu Gln Lys Arg 565 570 575 Asp His GluGly Phe Gly Phe Val Leu Arg Gly Ala Lys Ala Glu Thr 580 585 590 Pro IleGlu Glu Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr 595 600 605 LeuGlu Ser Val Asp Val Glu Gly Val Ala Trp Lys Ala Gly Leu Arg 610 615 620Thr Gly Asp Phe Leu Ile Glu Val Asn Gly Val Asn Val Val Lys Val 625 630635 640 Gly His Lys Gln Val Val Gly Leu Ile Arg Gln Gly Gly Asn Arg Leu645 650 655 Val Met Lys Val Val Ser Val Thr Arg Lys Pro Glu Glu Asp SerAla 660 665 670 Arg Arg Arg Ala Pro Pro Pro Pro Lys Arg Ala Pro Ser ThrThr Leu 675 680 685 Thr Leu Arg Ser Lys Ser Met Thr Ala Glu Leu Glu GluLeu Ala Ser 690 695 700 Ile Arg Arg Arg Lys Gly Glu Lys Leu Asp Glu IleLeu Ala Val Ala 705 710 715 720 Ala Glu Pro Thr Leu Arg Pro Asp Ile AlaAsp Ala Asp Ser Arg Ala 725 730 735 Ala Thr Val Lys Gln Arg Pro Thr SerArg Arg Ile Thr Pro Ala Glu 740 745 750 Ile Ser Ser Leu Phe Glu Arg GlnGly Leu Pro Gly Pro Glu Lys Leu 755 760 765 Pro Gly Ser Leu Arg Lys GlyIle Pro Arg Thr Lys Ser Val Gly Glu 770 775 780 Asp Glu Lys Leu Ala SerLeu Leu Glu Gly Arg Phe Pro Arg Ser Thr 785 790 795 800 Ser Met Gln AspThr Val Arg Glu Gly Arg Gly Ile Pro Pro Pro Pro 805 810 815 Gln Thr AlaPro Pro Pro Pro Pro Ala Pro Tyr Tyr Phe Asp Ser Gly 820 825 830 Pro ProPro Thr Phe Ser Pro Pro Pro Pro Pro Pro Gly Arg Ala Tyr 835 840 845 AspThr Val Arg Ser Ser Phe Lys Pro Gly Leu Glu Ala Arg Leu Gly 850 855 860Ala Gly Ala Ala Gly Leu Tyr Asp Ser Gly Thr Pro Leu Gly Pro Leu 865 870875 880 Pro Tyr Pro Glu Arg Gln Lys Arg Ala Arg Ser Met Ile Ile Leu Gln885 890 895 Asp Ser Ala Pro Glu Val Gly Asp Val Pro Arg Pro Ala Pro AlaAla 900 905 910 Thr Pro Pro Glu Arg Pro Lys Arg Arg Pro Arg Pro Ser GlyPro Asp 915 920 925 Ser Pro Tyr Ala Asn Leu Gly Ala Phe Ser Ala Ser LeuPhe Ala Pro 930 935 940 Ser Lys Pro Gln Arg Arg Lys Ser Pro Leu Val LysGln Leu Gln Val 945 950 955 960 Glu Asp Ala Gln Glu Arg Ala Ala Leu AlaVal Gly Ser Pro Gly Pro 965 970 975 Val Gly Gly Ser Phe Ala Arg Glu ProSer Pro Thr His Arg Gly Pro 980 985 990 Arg Pro Gly Gly Leu Asp Tyr SerSer Gly Glu Gly Leu Gly Leu Thr 995 1000 1005 Phe Gly Gly Pro Ser ProGly Pro Val Lys Glu Arg Arg Leu Glu 1010 1015 1020 Glu Arg Arg Arg SerThr Val Phe Leu Ser Val Gly Ala Ile Glu 1025 1030 1035 Gly Asn Pro ProSer Ala Asp Leu Pro Ser Leu Gln Pro Ser Arg 1040 1045 1050 Ser Ile AspGlu Arg Leu Leu Gly Thr Gly Ala Thr Thr Gly Arg 1055 1060 1065 Asp LeuLeu Leu Pro Ser Pro Val Ser Ala Leu Lys Pro Leu Val 1070 1075 1080 GlyGly Pro Asn Leu Gly Pro Ser Ser Ser Thr Phe Ile His Pro 1085 1090 1095Leu Thr Gly Lys Pro Leu Asp Pro Ser Ser Pro Leu Ala Leu Ala 1100 11051110 Leu Ala Ala Arg Glu Arg Ala Leu Ala Ser Gln Thr Pro Ser Arg 11151120 1125 Ser Pro Thr Pro Val His Ser Pro Asp Ala Asp Arg Pro Gly Pro1130 1135 1140 Leu Phe Val Asp Val Gln Thr Arg Asp Ser Glu Arg Gly ProLeu 1145 1150 1155 Ala Ser Pro Ala Phe Ser Pro Arg Ser Pro Ala Trp IlePro Val 1160 1165 1170 Pro Ala Arg Arg Glu Ala Glu Lys Pro Thr Arg GluGlu Arg Lys 1175 1180 1185 Ser Pro Glu Asp Lys Lys Ser Met Ile Leu SerVal Leu Asp Thr 1190 1195 1200 Ser Leu Gln Arg Pro Ala Gly Leu Ile ValVal His Ala Thr Ser 1205 1210 1215 Asn Gly Gln Glu Pro Asn Arg Leu GlyAla Glu Glu Glu Arg Pro 1220 1225 1230 Gly Thr Pro Glu Leu Ala Pro ThrPro Met Gln Ala Ala Ala Val 1235 1240 1245 Ala Glu Pro Met Pro Ser ProArg Ala Gln Pro Pro Gly Asn Ile 1250 1255 1260 Pro Ala Asp Pro Gly ProSer Gln Gly Asn Ser Glu Glu Glu Pro 1265 1270 1275 Lys Leu Val Phe AlaVal Asn Leu Pro Pro Ala Gln Leu Ser Ser 1280 1285 1290 Asn Asp Glu GluThr Arg Glu Glu Leu Ala Arg Ile Gly Leu Val 1295 1300 1305 Pro Pro ProGlu Glu Phe Ala Asn Gly Ile Leu Leu Ala Thr Pro 1310 1315 1320 Pro ProGly Pro Gly Pro Leu Pro Thr Thr Val Pro Ser Pro Ala 1325 1330 1335 SerGly Lys Pro Ser Ser Glu Leu Pro Pro Ala Pro Glu Ser Ala 1340 1345 1350Ala Asp Ser Gly Val Glu Glu Ala Asp Thr Arg Ser Ser Ser Asp 1355 13601365 Pro His Leu Glu Thr Thr Ser Thr Ile Ser Thr Val Ser Ser Met 13701375 1380 Ser Thr Leu Ser Ser Glu Ser Gly Glu Leu Thr Asp Thr His Thr1385 1390 1395 Ser Phe Ala Asp Gly His Thr Phe Leu Leu Glu Lys Pro ProVal 1400 1405 1410 Pro Pro Lys Pro Lys Leu Lys Ser Pro Leu Gly Lys GlyPro Val 1415 1420 1425 Thr Phe Arg Gly Pro Leu Leu Lys Gln Ser Ser AspSer Glu Leu 1430 1435 1440 Met Ala Gln Gln His His Ala Thr Ser Thr GlyLeu Thr Ser Ala 1445 1450 1455 Ala Gly Pro Ala Arg Pro Arg Tyr Leu PheGln Arg Arg Ser Lys 1460 1465 1470 Leu Trp Gly Asp Pro Val Glu Ser ArgGly Leu Pro Gly Pro Glu 1475 1480 1485 Asp Asp Lys Pro Thr Val Ile SerGlu Leu Ser Ser Arg Leu Gln 1490 1495 1500 Gln Leu Asn Lys Asp Thr ArgSer Leu Gly Glu Glu Pro Val Gly 1505 1510 1515 Gly Leu Gly Ser Leu LeuAsp Pro Ala Lys Lys Ser Pro Ile Ala 1520 1525 1530 Ala Ala Arg Cys AlaVal Val Pro Ser Ala Gly Trp Leu Phe Ser 1535 1540 1545 Ser Leu Gly GluLeu Ser Thr Ile Ser Ala Gln Arg Ser Pro Gly 1550 1555 1560 Gly Pro GlyGly Gly Ala Ser Tyr Ser Val Arg Pro Ser Gly Arg 1565 1570 1575 Tyr ProVal Ala Arg Arg Ala Pro Ser Pro Val Lys Pro Ala Ser 1580 1585 1590 LeuGlu Arg Val Glu Gly Leu Gly Ala Gly Val Gly Gly Ala Gly 1595 1600 1605Arg Pro Phe Gly Leu Thr Pro Pro Thr Ile Leu Lys Ser Ser Ser 1610 16151620 Leu Ser Ile Pro His Glu Pro Lys Glu Val Arg Phe Val Val Arg 16251630 1635 Ser Ala Ser Ala Arg Ser Arg Ser Pro Ser Pro Ser Pro Leu Pro1640 1645 1650 Ser Pro Ser Pro Gly Ser Gly Pro Ser Ala Gly Pro Arg ArgPro 1655 1660 1665 Phe Gln Gln Lys Pro Leu Gln Leu Trp Ser Lys Phe AspVal Gly 1670 1675 1680 Asp Trp Leu Glu Ser Ile His Leu Gly Glu His ArgAsp Arg Phe 1685 1690 1695 Glu Asp His Glu Ile Glu Gly Ala His Leu ProAla Leu Thr Lys 1700 1705 1710 Glu Asp Phe Val Glu Leu Gly Val Thr ArgVal Gly His Arg Met 1715 1720 1725 Asn Ile Glu Arg Ala Leu Arg Gln LeuAsp Gly Ser 1730 1735 1740 <210> SEQ ID NO 41 <211> LENGTH: 474 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(435) <400> SEQUENCE: 41 atg tcc aca gcc agggag cag cca atc ttc agc aca cgg gcg cac gtg 48 Met Ser Thr Ala Arg GluGln Pro Ile Phe Ser Thr Arg Ala His Val 1 5 10 15 ttc caa att gac ccagcc acc aag cga aac tgg atc cca gcg ggc aag 96 Phe Gln Ile Asp Pro AlaThr Lys Arg Asn Trp Ile Pro Ala Gly Lys 20 25 30 cac gca ctc act gtc tcctat ttc tac gat gcc acc cgc aat gtg tac 144 His Ala Leu Thr Val Ser TyrPhe Tyr Asp Ala Thr Arg Asn Val Tyr 35 40 45 cgc atc atc agc atc gga ggcgcc aag gcc atc atc aac agc act gtc 192 Arg Ile Ile Ser Ile Gly Gly AlaLys Ala Ile Ile Asn Ser Thr Val 50 55 60 act ccc aac atg acc ttc acc aaaact tcc cag aag ttc ggg cag tgg 240 Thr Pro Asn Met Thr Phe Thr Lys ThrSer Gln Lys Phe Gly Gln Trp 65 70 75 80 gcc gac agt cgc gcc aac aca gtctat ggc ctg ggc ttt gcc tct gaa 288 Ala Asp Ser Arg Ala Asn Thr Val TyrGly Leu Gly Phe Ala Ser Glu 85 90 95 cag cat ctg aca cag ttt gcc gag aagttc cag gaa gtg aag gaa gca 336 Gln His Leu Thr Gln Phe Ala Glu Lys PheGln Glu Val Lys Glu Ala 100 105 110 gcc agg ctg gcc agg gag aaa tct caggat ggc tgg ggt ggg ccc cag 384 Ala Arg Leu Ala Arg Glu Lys Ser Gln AspGly Trp Gly Gly Pro Gln 115 120 125 tcg gct ctg gtt gtt ggc agc ttt ggggct gtt ttt gag ctt ctc att 432 Ser Ala Leu Val Val Gly Ser Phe Gly AlaVal Phe Glu Leu Leu Ile 130 135 140 gtg tagaatttct agatcccccg attacatttctaagcgtga 474 Val 145 <210> SEQ ID NO 42 <211> LENGTH: 145 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42 Met Ser Thr Ala ArgGlu Gln Pro Ile Phe Ser Thr Arg Ala His Val 1 5 10 15 Phe Gln Ile AspPro Ala Thr Lys Arg Asn Trp Ile Pro Ala Gly Lys 20 25 30 His Ala Leu ThrVal Ser Tyr Phe Tyr Asp Ala Thr Arg Asn Val Tyr 35 40 45 Arg Ile Ile SerIle Gly Gly Ala Lys Ala Ile Ile Asn Ser Thr Val 50 55 60 Thr Pro Asn MetThr Phe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp 65 70 75 80 Ala Asp SerArg Ala Asn Thr Val Tyr Gly Leu Gly Phe Ala Ser Glu 85 90 95 Gln His LeuThr Gln Phe Ala Glu Lys Phe Gln Glu Val Lys Glu Ala 100 105 110 Ala ArgLeu Ala Arg Glu Lys Ser Gln Asp Gly Trp Gly Gly Pro Gln 115 120 125 SerAla Leu Val Val Gly Ser Phe Gly Ala Val Phe Glu Leu Leu Ile 130 135 140Val 145 <210> SEQ ID NO 43 <211> LENGTH: 859 <212> TYPE: DNA <213>ORGANISM: Rattus norvegicus <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(858) <400> SEQUENCE: 43 cac gcg tcc ggt gtg gtg cac cttggc atc tgt aag cct ttg gtg gag 48 His Ala Ser Gly Val Val His Leu GlyIle Cys Lys Pro Leu Val Glu 1 5 10 15 gag gag aag gag gag aag gag gaacat ttt att ttc cat tca aac aac 96 Glu Glu Lys Glu Glu Lys Glu Glu HisPhe Ile Phe His Ser Asn Asn 20 25 30 aat gga gat aac agt gag tct cca gaaacc gtt cac gag atc cac tca 144 Asn Gly Asp Asn Ser Glu Ser Pro Glu ThrVal His Glu Ile His Ser 35 40 45 tct tta atc ctc gag gca ccc cag gga tttaga gat gag ccg tat ctt 192 Ser Leu Ile Leu Glu Ala Pro Gln Gly Phe ArgAsp Glu Pro Tyr Leu 50 55 60 gaa gaa ctc gtg gat gaa cct ttt cta gat ttggga aag tct ttg cag 240 Glu Glu Leu Val Asp Glu Pro Phe Leu Asp Leu GlyLys Ser Leu Gln 65 70 75 80 ttc caa caa aaa gac atg gac agc agc tca gaagcc tgg gaa atg cat 288 Phe Gln Gln Lys Asp Met Asp Ser Ser Ser Glu AlaTrp Glu Met His 85 90 95 gaa ttc ctg agc cct cgg ctg gag aga agg ggt gaggaa aga gag atg 336 Glu Phe Leu Ser Pro Arg Leu Glu Arg Arg Gly Glu GluArg Glu Met 100 105 110 ctt gtt gac gag gag tat gag atc tac caa gac cgcctc cgg gac atg 384 Leu Val Asp Glu Glu Tyr Glu Ile Tyr Gln Asp Arg LeuArg Asp Met 115 120 125 gaa gca cac cca cca cct cct cac att cgg gag cccact tct gca tct 432 Glu Ala His Pro Pro Pro Pro His Ile Arg Glu Pro ThrSer Ala Ser 130 135 140 ccc agg ctg gat ctc cag gcc ggc ccc cag tgg ctgcat gct gac ctc 480 Pro Arg Leu Asp Leu Gln Ala Gly Pro Gln Trp Leu HisAla Asp Leu 145 150 155 160 tca gga gga gag ata ctc gag tgt cac gac acagag tcc atg atg act 528 Ser Gly Gly Glu Ile Leu Glu Cys His Asp Thr GluSer Met Met Thr 165 170 175 gct tat ccc cag gag atg cag gac tat agc ttcagc acc aca gac atg 576 Ala Tyr Pro Gln Glu Met Gln Asp Tyr Ser Phe SerThr Thr Asp Met 180 185 190 atg aaa gaa aca ttt ggc ctt gac tcc cgg ccgccc atg ccc tcc tct 624 Met Lys Glu Thr Phe Gly Leu Asp Ser Arg Pro ProMet Pro Ser Ser 195 200 205 gaa gga aat ggt cag cac ggc cga ttt gat gacttg gaa cat ctt cat 672 Glu Gly Asn Gly Gln His Gly Arg Phe Asp Asp LeuGlu His Leu His 210 215 220 tca cta gca agc cac ggc ctg gat tta ggc atgatg act cca agt gac 720 Ser Leu Ala Ser His Gly Leu Asp Leu Gly Met MetThr Pro Ser Asp 225 230 235 240 ttg caa ggc cct ggc gtg ctt gta gat cttcca gct gtc acc cca aga 768 Leu Gln Gly Pro Gly Val Leu Val Asp Leu ProAla Val Thr Pro Arg 245 250 255 aga ggc tgc ggc cgc taa gta agt aag acgtcg agc tct aag taa gta 816 Arg Gly Cys Gly Arg Val Ser Lys Thr Ser SerSer Lys Val 260 265 270 acg gcc gcc acc gcg gtg gag ctt tgg act tct tcgcca gag g 859 Thr Ala Ala Thr Ala Val Glu Leu Trp Thr Ser Ser Pro Glu275 280 <210> SEQ ID NO 44 <211> LENGTH: 261 <212> TYPE: PRT <213>ORGANISM: Rattus norvegicus <400> SEQUENCE: 44 His Ala Ser Gly Val ValHis Leu Gly Ile Cys Lys Pro Leu Val Glu 1 5 10 15 Glu Glu Lys Glu GluLys Glu Glu His Phe Ile Phe His Ser Asn Asn 20 25 30 Asn Gly Asp Asn SerGlu Ser Pro Glu Thr Val His Glu Ile His Ser 35 40 45 Ser Leu Ile Leu GluAla Pro Gln Gly Phe Arg Asp Glu Pro Tyr Leu 50 55 60 Glu Glu Leu Val AspGlu Pro Phe Leu Asp Leu Gly Lys Ser Leu Gln 65 70 75 80 Phe Gln Gln LysAsp Met Asp Ser Ser Ser Glu Ala Trp Glu Met His 85 90 95 Glu Phe Leu SerPro Arg Leu Glu Arg Arg Gly Glu Glu Arg Glu Met 100 105 110 Leu Val AspGlu Glu Tyr Glu Ile Tyr Gln Asp Arg Leu Arg Asp Met 115 120 125 Glu AlaHis Pro Pro Pro Pro His Ile Arg Glu Pro Thr Ser Ala Ser 130 135 140 ProArg Leu Asp Leu Gln Ala Gly Pro Gln Trp Leu His Ala Asp Leu 145 150 155160 Ser Gly Gly Glu Ile Leu Glu Cys His Asp Thr Glu Ser Met Met Thr 165170 175 Ala Tyr Pro Gln Glu Met Gln Asp Tyr Ser Phe Ser Thr Thr Asp Met180 185 190 Met Lys Glu Thr Phe Gly Leu Asp Ser Arg Pro Pro Met Pro SerSer 195 200 205 Glu Gly Asn Gly Gln His Gly Arg Phe Asp Asp Leu Glu HisLeu His 210 215 220 Ser Leu Ala Ser His Gly Leu Asp Leu Gly Met Met ThrPro Ser Asp 225 230 235 240 Leu Gln Gly Pro Gly Val Leu Val Asp Leu ProAla Val Thr Pro Arg 245 250 255 Arg Gly Cys Gly Arg 260 <210> SEQ ID NO45 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Rattus norvegicus<400> SEQUENCE: 45 Val Ser Lys Thr Ser Ser Ser Lys 1 5 <210> SEQ ID NO46 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Rattus norvegicus<400> SEQUENCE: 46 Val Thr Ala Ala Thr Ala Val Glu Leu Trp Thr Ser SerPro Glu 1 5 10 15 <210> SEQ ID NO 47 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: optimal ligand <400> SEQUENCE: 47 Phe Pro Pro Pro Pro 1 5<210> SEQ ID NO 48 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: motif ligand<400> SEQUENCE: 48 Lys Ile Ala Ala 1 <210> SEQ ID NO 49 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: oligonucleotide for PCR <400> SEQUENCE: 49 gacagcagagccaacaccgt g 21 <210> SEQ ID NO 50 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: oligonucleotide for PCR <400> SEQUENCE: 50 gtctgcagctccatctccca c 21 <210> SEQ ID NO 51 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: oligonucleotide for PCR <400> SEQUENCE: 51 cacggtgttggctctgctgt c 21 <210> SEQ ID NO 52 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: oligonucleotide for PCR <400> SEQUENCE: 52 atgggvgarcarccbatytt c 21 <210> SEQ ID NO 53 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:conserved amino acid sequence <400> SEQUENCE: 53 Met Gly Glu Gln Pro IlePhe 1 5 <210> SEQ ID NO 54 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:oligonucleotide for PCR <400> SEQUENCE: 54 gagggtagcc agttcagcct c 21<210> SEQ ID NO 55 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:oligonucleotide for PCR <400> SEQUENCE: 55 gttgatctca ctgcattgtt c 21<210> SEQ ID NO 56 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: peptide fromHomer 1b/c <400> SEQUENCE: 56 Ile Phe Glu Leu Thr Glu Leu Arg Asp AsnLeu Ala Lys Leu Leu Glu 1 5 10 15 Cys Ser <210> SEQ ID NO 57 <211>LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: peptide from Homer 2a/b <400>SEQUENCE: 57 Gly Lys Ile Asp Asp Leu His Asp Phe Arg Arg Gly Leu Ser LysLeu 1 5 10 15 Gly Thr Asp Asn 20 <210> SEQ ID NO 58 <211> LENGTH: 19<212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: peptide from Homer 3 <400> SEQUENCE: 58 Arg Leu PheGlu Leu Ser Glu Leu Arg Glu Gly Leu Ala Arg Leu Ala 1 5 10 15 Glu AlaAla <210> SEQ ID NO 59 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: peptide withHomer ligand peptide consensus <400> SEQUENCE: 59 Leu Val Pro Pro ProGlu Glu Phe Ala Asn 1 5 10 <210> SEQ ID NO 60 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: peptide with Homer ligand peptide consensus <400> SEQUENCE:60 Pro Leu Pro Pro Pro Leu Glu Phe Ser Asn 1 5 10 <210> SEQ ID NO 61<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: peptide with Homer ligandpeptide consensus <400> SEQUENCE: 61 Pro Leu Pro Pro Pro Leu Glu Phe AlaAsn 1 5 10 <210> SEQ ID NO 62 <211> LENGTH: 10 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:peptide with Homer ligand peptide consensus <400> SEQUENCE: 62 Phe LeuPro Pro Pro Glu Ser Phe Asp Ala 1 5 10 <210> SEQ ID NO 63 <211> LENGTH:113 <212> TYPE: PRT <213> ORGANISM: Rat <400> SEQUENCE: 63 Met Gly GluGln Pro Ile Phe Ser Thr Arg Ala His Val Phe Gln Ile 1 5 10 15 Asp ProAsn Thr Lys Lys Asn Trp Val Pro Thr Ser Lys His Ala Val 20 25 30 Thr ValSer Tyr Phe Tyr Asp Ser Thr Arg Asn Val Tyr Arg Ile Ile 35 40 45 Ser LeuAsp Gly Ser Lys Ala Ile Ile Asn Ser Thr Ile Thr Pro Asn 50 55 60 Met ThrPhe Thr Lys Thr Ser Gln Lys Phe Gly Gln Trp Ala Asp Ser 65 70 75 80 ArgAla Asn Thr Val Tyr Gly Leu Gly Phe Ser Ser Glu His His Leu 85 90 95 SerLys Phe Ala Glu Lys Phe Gln Glu Phe Lys Glu Ala Ala Arg Leu 100 105 110Ala <210> SEQ ID NO 64 <211> LENGTH: 113 <212> TYPE: PRT <213> ORGANISM:Drosophila <400> SEQUENCE: 64 Met Gly Glu Gln Pro Ile Phe Thr Cys GlnAla His Val Phe His Ile 1 5 10 15 Asp Pro Lys Thr Lys Arg Thr Trp IleThr Ala Ser Met Lys Ala Val 20 25 30 Asn Val Ser Phe Phe Tyr Asp Ser SerArg Asn Leu Tyr Arg Ile Ile 35 40 45 Ser Val Glu Gly Thr Lys Ala Val IleAsn Ser Thr Ile Thr Pro Asn 50 55 60 Met Thr Phe Thr Gln Thr Ser Gln LysPhe Gly Gln Trp Ser Asp Val 65 70 75 80 Arg Ala Asn Thr Val Tyr Gly LeuGly Phe Ala Ser Glu Ala Glu Ile 85 90 95 Thr Lys Phe Val Glu Lys Phe GlnGlu Val Lys Glu Ala Thr Lys Asn 100 105 110 Ala <210> SEQ ID NO 65 <211>LENGTH: 113 <212> TYPE: PRT <213> ORGANISM: Drosophila <400> SEQUENCE:65 Met Thr Glu Gln Ser Ile Ile Gly Ala Arg Ala Ser Val Met Val Tyr 1 510 15 Asp Asp Asn Gln Lys Lys Trp Val Pro Ser Gly Ser Ser Ser Gly Leu 2025 30 Ser Lys Val Gln Ile Tyr His His Gln Gln Asn Asn Thr Phe Arg Val 3540 45 Val Gly Arg Lys Leu Gln Asp His Glu Val Val Ile Asn Cys Ser Ile 5055 60 Leu Lys Gly Leu Lys Tyr Asn Gln Ala Thr Ala Thr Phe His Gln Trp 6570 75 80 Arg Asp Ser Lys Phe Val Tyr Gly Leu Asn Phe Ser Ser Gln Asn Ala85 90 95 Glu Asn Phe Ala Arg Ala Met Met His Ala Leu Glu Val Leu Ser Gly100 105 110 Arg <210> SEQ ID NO 66 <211> LENGTH: 114 <212> TYPE: PRT<213> ORGANISM: Mouse <400> SEQUENCE: 66 Met Ser Glu Gln Ser Ile Cys GlnAla Arg Ala Ala Val Met Val Tyr 1 5 10 15 Asp Asp Ala Asn Lys Lys TrpVal Pro Ala Gly Gly Ser Thr Gly Phe 20 25 30 Ser Arg Val His Ile Tyr HisHis Thr Gly Asn Asn Thr Phe Arg Val 35 40 45 Val Gly Arg Lys Ile Gln AspHis Gln Val Val Ile Asn Cys Ala Ile 50 55 60 Pro Lys Gly Leu Lys Tyr AsnGln Ala Thr Gln Thr Phe His Gln Trp 65 70 75 80 Arg Asp Ala Arg Gln ValTyr Gly Leu Asn Phe Gly Ser Lys Glu Asp 85 90 95 Ala Asn Val Phe Ala SerAla Met Met His Ala Leu Glu Val Leu Asn 100 105 110 Ser Gln <210> SEQ IDNO 67 <211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM: Mouse <400>SEQUENCE: 67 Met Ser Glu Gln Ser Ile Cys Gln Ala Arg Ala Ser Val Met ValTyr 1 5 10 15 Asp Asp Thr Ser Lys Lys Trp Val Pro Ile Lys Pro Gly GlnGln Gly 20 25 30 Phe Ser Arg Ile Asn Ile Tyr His Asn Thr Ala Ser Ser ThrPhe Arg 35 40 45 Val Val Gly Val Lys Leu Gln Asp Gln Gln Val Val Ile AsnTyr Ser 50 55 60 Ile Val Lys Gly Leu Lys Tyr Asn Gln Ala Thr Pro Thr PheHis Gln 65 70 75 80 Trp Arg Asp Ala Arg Gln Val Tyr Gly Leu Asn Phe AlaSer Lys Glu 85 90 95 Glu Ala Thr Thr Phe Ser Asn Ala Met Leu Phe Ala LeuAsn Ile Met 100 105 110 Asn Ser Gln 115 <210> SEQ ID NO 68 <211> LENGTH:119 <212> TYPE: PRT <213> ORGANISM: Drosophila <400> SEQUENCE: 68 GlyHis Leu Leu Ser Ser Phe Arg Leu Trp Ala Glu Val Phe His Val 1 5 10 15Ser Ala Ser Gly Ala Gly Thr Val Lys Trp Gln Gln Val Ser Glu Asp 20 25 30Leu Val Pro Val Asn Ile Thr Cys Ile Gln Asp Ser Pro Glu Cys Ile 35 40 45Phe His Ile Thr Ala Tyr Asn Ser Gln Val Asp Lys Ile Leu Asp Val 50 55 60Arg Leu Val Gln Pro Gly Thr Arg Ile Gly Gln Ala Ser Glu Cys Phe 65 70 7580 Val Tyr Trp Lys Asp Pro Met Thr Asn Asp Thr Trp Gly Leu Asn Phe 85 9095 Thr Ser Pro Ile Asp Ala Lys Gln Phe Arg Glu Cys Cys Ser Pro Ser 100105 110 Phe Lys Phe Ser Arg Lys Ala 115 <210> SEQ ID NO 69 <211> LENGTH:116 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 69 MetSer Glu Thr Val Ile Cys Ser Ser Arg Ala Thr Val Met Leu Tyr 1 5 10 15Asp Asp Gly Asn Lys Arg Trp Leu Pro Ala Gly Thr Gly Pro Gln Ala 20 25 30Phe Ser Arg Val Gln Ile Tyr His Asn Pro Thr Ala Asn Ser Phe Arg 35 40 45Val Val Gly Arg Lys Met Gln Pro Asp Gln Gln Val Val Ile Asn Cys 50 55 60Ala Ile Val Arg Gly Val Lys Tyr Asn Gln Ala Thr Pro Asn Phe His 65 70 7580 Gln Trp Arg Asp Ala Arg Gln Val Trp Gly Leu Asn Phe Gly Ser Lys 85 9095 Glu Asp Ala Ala Gln Phe Ala Ala Gly Met Ala Ser Ala Leu Glu Ala 100105 110 Leu Glu Gly Gly 115 <210> SEQ ID NO 70 <211> LENGTH: 110 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 70 Phe Leu GlyLys Lys Cys Val Thr Met Ser Ser Ala Val Val Gln Leu 1 5 10 15 Tyr AlaAla Asp Arg Asn Cys Met Trp Ser Lys Lys Cys Ser Gly Val 20 25 30 Ala CysLeu Val Lys Asp Asn Pro Gln Arg Ser His Phe Ile Arg Ile 35 40 45 Phe AspIle Lys Asp Gly Lys Leu Trp Glu Gln Glu Leu Tyr Asn Asn 50 55 60 Phe ValTyr Asn Ser Pro Arg Gly Tyr Phe His Thr Phe Ala Gly Asp 65 70 75 80 ThrCys Gln Val Ala Leu Asn Phe Ala Asn Glu Glu Glu Ala Lys Lys 85 90 95 PheArg Lys Ala Val Thr Asp Leu Leu Gly Arg Arg Gln Arg 100 105 110 <210>SEQ ID NO 71 <211> LENGTH: 114 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 71 Met Leu Gly Arg Lys Cys Leu Thr Leu Ala ThrAla Val Val Gln Leu 1 5 10 15 Tyr Leu Ala Leu Pro Pro Gly Ala Glu HisTrp Thr Lys Glu His Cys 20 25 30 Gly Ala Val Cys Leu Val Lys Asp Asn ProGln Lys Ser Tyr Phe Ile 35 40 45 Arg Leu Tyr Gly Leu Gln Ala Gly Arg LeuLeu Trp Glu Gln Glu Leu 50 55 60 Tyr Ser Gln Leu Val Tyr Ser Thr Pro ThrPro Phe Phe His Thr Phe 65 70 75 80 Ala Gly Asp Asp Cys Gln Ala Gly LeuAsn Phe Ala Asp Glu Asp Glu 85 90 95 Ala Gln Ala Phe Arg Ala Leu Val GlnGlu Lys Ile Gln Lys Arg Asn 100 105 110 Gln Arg <210> SEQ ID NO 72 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Mutations in the EVH1 domain of theWASP gene <400> SEQUENCE: 72 Pro Trp Met Pro Asp Ile Glu Val Pro Met AspCys Arg Ser Lys Lys 1 5 10 15

What is claimed is:
 1. An isolated nucleic acid encoding Homer protein1b, wherein said nucleic acid has the nucleotide sequence set forth inSEQ ID NO:3.
 2. An expression vector encoding a polynucleotide ofclaim
 1. 3. The expression vector of claim 2, wherein the vector isvirus-derived.
 4. The expression vector of claim 2, wherein the vectoris a plasmid.
 5. A host cell comprising a vector of claim
 2. 6. The hostcell of claim 5, wherein the host cell is a prokaryotic cell.
 7. Thehost cell of claim 5, wherein the host cell is a eukaryotic cell.